Hypersensitive response elicitor-derived peptides and use thereof

ABSTRACT

Disclosed are hypersensitive-response eliciting peptides and non-hypersensitive response eliciting peptides that induce active plant responses, and that exhibit improved solubility, stability, resistance to chemical degradation, or a combination of these properties. Use of these peptides or fusion polypeptides, or DNA constructs encoding the same, for modulating plant biochemical signaling, imparting disease resistance to plants, enhancing plant growth, imparting tolerance to biotic stress, imparting tolerance and resistance to abiotic stress, imparting desiccation resistance to cuttings removed from ornamental plants, imparting post-harvest disease or post-harvest desiccation resistance to a fruit or vegetable, or enhancing the longevity of fruit or vegetable ripeness are also disclosed.

This application claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 62/319,138, filed Apr. 6, 2016, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel hypersensitive response elicitorpeptides and their use for inducing active plant responses including,among others, growth enhancement, disease resistance, pest or insectresistance, and stress resistance.

BACKGROUND OF THE INVENTION

The identification and isolation of harpin proteins came from basicresearch at Cornell University attempting to understand how plantpathogenic bacteria interact with plants. A first line of defense is thehypersensitive response (HR), a localized plant cell death at the siteof infection. Cell death creates a physical barrier to movement of thepathogen and in some plants dead cells can release compounds toxic tothe invading pathogen. Research had indicated that pathogenic bacteriawere likely to have a single factor that was responsible for triggeringthe HR. A basic aim of the Cornell research was to identify a specificbacterial protein responsible for eliciting the HR. The target proteinwas known to be encoded by one of a group of bacteria genes called theHypersensitive Response and Pathogenicity (hrp) gene cluster. The hrpcluster in the bacterium Erwinia amylovora (Ea), which causes fireblight in pear and apple, was dissected and a single protein wasidentified that elicited HR in certain plants. This protein was giventhe name harpin (and, later, harpin_(Ea) and the corresponding genedesignated hrpN. This was the first example of such a protein and geneidentified from any bacterial species.

A number of different harpin proteins have since been identified fromErwinia, Pseudomonas, Ralstonia, Xanthomonas, and Pantoea species, amongothers. Harpin proteins, while diverse at the primary amino acidsequence level, share common biochemical and biophysical characteristicsas well as biological functions. Based on their unique properties, theharpin proteins are regarded in the literature as belonging to a singleclass of proteins.

Subsequent to their identification and isolation, it was thereafterdiscovered that harpins could elicit disease resistance in plants andincrease plant growth. An important early finding was that applicationof purified harpin protein made a plant resistant to a subsequentpathogen attack, and in locations on the plant well away from theinjection site. This meant that harpin proteins can trigger a SystemicAcquired Resistance (SAR), a plant defense mechanism that providesresistance to a variety of viral, bacterial, and fungal pathogens.

In crop protection, there is a continuous need for compositions thatimprove the health of plants. Healthier plants are desirable since theyresult in better yields and/or a better quality of the plants or crops.Healthier plants also better resist biotic and abiotic stress. A highresistance against biotic stresses in turn allows the growers to reducethe quantity of pesticides applied and consequently to slow down thedevelopment of resistances against the respective pesticides.

Harpin_(αβ) is a fusion protein that is derived from several differentharpins. Harpin_(αβ) has been shown to suppress nematode egg production,enhance the growth, quality and yield of a plant, and increase a plant'svigor. Its amino acid and nucleotide sequences are described in detailin U.S. Application Publ. No. 2010/0043095.

To date, harpin and harpin_(αβ) production and their use in agriculturaland horticultural applications have been as a powdered solid coated onstarch. This limits the use and versatility of the harpin proteins,because liquid suspensions of the powdered harpin proteins in water havean effective useful life of only 48-72 hours before significantdegradation and loss of activity occurs. Another problem with harpinsolutions is protein solubility and stability.

It would be desirable to identify synthetic and derivative harpinpeptides that are readily soluble in aqueous solution, stable, resistantto chemical degradation, and effective in initiating one or more activeplant responses including, without limitation, the hypersensitive plantresponse.

The present invention is directed to overcoming these and otherlimitations in the art.

SUMMARY OF THE INVENTION

A first aspect of the invention relates to an isolated peptidecomprising the amino acid sequence of

(i)  (SEQ ID NO: 1) (L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X-(L/I/V/F),  or (ii)  (SEQ ID NO: 2)L-X-X-L-L-L-X-(F/L)-(I/L)-X-X-X-L,wherein for both SEQ ID NO: 1 and 2, X at position 3 is optional and,when present, is any amino acid; and each X at positions 2, 7, 10, 11,and 12 is any amino acid. In one embodiment, X at position 3 is present.In another embodiment, X at position 3 is not present. In otherembodiments, the isolated peptide is free of cysteine or free of bothcysteine and methionine. In certain embodiments, the isolated peptidefurther includes a hydrophilic amino acid sequence that is locatedN-terminal or C-terminal to SEQ ID NO: 1 or SEQ ID NO: 2.

One set of peptides according to the first aspect of the invention havethe amino acid sequence of:

(SEQ ID NO: 3) (Q/E)-(Q/E)-(L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X-(L/I/V/F)- (D/G/Q/E)-(D/G/Q/E),,wherein X at position 5 is optional and, when present, is any aminoacid; and each X at positions 4, 9, 12, 13, and 14 is any amino acid. Inone embodiment, X at position 5 is present. In another embodiment, X atposition 5 is not present.

Another set of peptides according to the first aspect of the inventionhave the amino acid of:

(SEQ ID NO: 4) (L/I/V/F)-X-X-(L/I/V/F)-(L/IN/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X-(L/I/V/F)-(D/G/Q/E)- (D/G/Q/E),,wherein X at position 3 is optional and, when present, is any aminoacid; and each X at positions 2, 7, 10, 11, and 12 is any amino acid. Inone embodiment, X at position 3 is present. In another embodiment, X atposition 3 is not present.

A further set of peptides according to the first aspect of the inventionhave the amino acid sequence of:

(SEQ ID NO: 5) (Q/E)-(Q/E)-(L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X-(L/I/V/F),,wherein X at position 5 is optional and, when present, is any aminoacid; and each X at positions 4, 9, 12, 13, and 14 is any amino acid. Inone embodiment, X at position 5 is present. In another embodiment, X atposition 5 is not present.

A second aspect of the invention relates to an isolated peptidecomprising the amino acid sequence of

(SEQ ID NO: 6) J-X-X-J-J-J-X-J-J-X-X-X-J wherein X at position 3 is optional and, when present, is any aminoacid; and each X at positions 2, 7, 10, 11, and 12 is any amino acid;one to three of the J residues at positions 1, 4, 5, 6, 8, 9, and 13 isa non-hydrophobic amino acid or A, and all other of the J residues areL, I, V, or F, and the peptide induces an active plant response, butdoes not induce a hypersensitive response, when applied to plant tissue.

A third aspect of the invention relates to a fusion protein thatincludes one of the peptides of the first or second aspect of theinvention along with one or more of a purification tag, a solubilitytag, or a second peptide according to the first or second aspect of theinvention.

A fourth aspect of the invention relates to a composition that includesone or more peptides according to the first or second aspect of theinvention, or a fusion protein according to the third aspect of theinvention, and a carrier.

A fifth aspect of the invention relates to a method of imparting diseaseresistance to plants. This method includes: applying an effective amountof an isolated peptide according to the first or second aspect of theinvention, a fusion protein according to the third aspect of theinvention, or a composition according to the fourth aspect of theinvention to a plant or plant seed or the locus where the plant isgrowing or is expected to grow, wherein said applying is effective toimpart disease resistance.

A sixth aspect of the invention relates to a method of enhancing plantgrowth. This method includes: applying an effective amount of anisolated peptide according to the first or second aspect of theinvention, a fusion protein according to the third aspect of theinvention, or a composition according to the fourth aspect of theinvention to a plant or plant seed or the locus where the plant isgrowing or is expected to grow, wherein said applying is effective toenhance plant growth.

A seventh aspect of the invention relates to a method of increasing aplant's tolerance and resistance to biotic stressors. This methodincludes: applying an effective amount of an isolated peptide accordingto the first or second aspect of the invention, a fusion proteinaccording to the third aspect of the invention, or a compositionaccording to the fourth aspect of the invention to a plant or plant seedor the locus where the plant is growing or is expected to grow, whereinsaid applying is effective to increase the plant's tolerance andresistance to biotic stress factors selected from the group consistingof pests such as insects, arachnids, nematodes, weeds, and combinationsthereof.

An eighth aspect of the invention relates to a method of increasing aplant's tolerance to abiotic stress. This method includes: applying aneffective amount of an isolated peptide according to the first or secondaspect of the invention, a fusion protein according to the third aspectof the invention, or a composition according to the fourth aspect of theinvention to a plant or plant seed or the locus where the plant isgrowing or is expected to grow, wherein said applying is effective toincrease the plant's tolerance to abiotic stress factors selected fromthe group consisting of salt stress, water stress (including drought andflooding), ozone stress, heavy metal stress, cold stress, heat stress,nutritional stress (phosphate, potassium, nitrogen deficiency),bleaching and light-induced stress, and combinations thereof.

A ninth aspect of the invention relates to a method impartingdesiccation resistance to cuttings removed from ornamental plants. Thismethod includes: applying an isolated peptide according to the first orsecond aspect of the invention, a fusion protein according to the thirdaspect of the invention, or a composition according to the fourth aspectof the invention to a plant or the locus where the plant is growing,wherein said applying is effective to impart desiccation resistance tocuttings removed from the ornamental plant.

A tenth aspect of the invention relates to a method of impartingpost-harvest disease or post-harvest desiccation resistance to a fruitor vegetable. This method includes: applying an effective amount of anisolated peptide according to the first or second aspect of theinvention, a fusion protein according to the third aspect of theinvention, or a composition according to the fourth aspect of theinvention to a plant containing a fruit or vegetable or the locus wherethe plant is growing; or applying an effective amount of the isolatedpeptide, the fusion protein, or the composition to a harvested fruit orvegetable, wherein said applying is effective to impart post-harvestdisease resistance or desiccation resistance to the fruit or vegetable.

An eleventh aspect of the invention relates to a method of enhancing thelongevity of fruit or vegetable ripeness. This method includes: applyingan effective amount of an isolated peptide according to the first orsecond aspect of the invention, a fusion protein according to the thirdaspect of the invention, or a composition according to the fourth aspectof the invention to a plant containing a fruit or vegetable or the locuswhere the plant is growing; or applying an effective amount of theisolated peptide, the fusion protein, or the composition to a harvestedfruit or vegetable, wherein said applying is effective to enhance thelongevity of fruit or vegetable ripeness.

A twelfth aspect of the invention relates to a method of modulating oneor more biological signaling processes of a plant. This method includes:applying an effective amount of an isolated peptide according to thefirst or second aspect of the invention, a fusion protein according tothe third aspect of the invention, or a composition according to thefourth aspect of the invention to a plant or the locus where the plantis growing, wherein said applying is effective in modulating one or morebiochemical signaling processes.

A thirteenth aspect of the invention relates to a DNA constructincluding a first nucleic acid molecule encoding a polypeptide accordingto the first or second aspect of the invention or a fusion proteinaccording to the third aspect of the invention; and a promoter-effectivenucleic acid molecule operably coupled to the first nucleic acidmolecule. This aspect of the invention also encompasses a recombinantexpression vector containing the DNA construct, a recombinant host cellcontaining the DNA construct, as well as transgenic plants or plantseeds that include a recombinant plant cell of the invention (whichcontains the DNA construct).

A fourteenth aspect of the invention relates to a method of impartingdisease resistance to plants, enhancing plant growth, impartingtolerance and resistance to biotic stressors, imparting tolerance toabiotic stress, or modulating plant biochemical signaling. This methodincludes providing a transgenic plant transformed with a DNA constructaccording to the thirteenth aspect of the invention; and growing theplant under conditions effective to permit the DNA construct to expressthe peptide or the fusion polypeptide to impart disease resistance,enhance plant growth, impart tolerance to biotic stress, imparttolerance to abiotic stress, or modulate biochemical signaling to thetransgenic plant.

A fifteenth aspect of the invention relates to a method of impartingdesiccation resistance to cuttings removed from ornamental plants,imparting post-harvest disease or post-harvest desiccation resistance toa fruit or vegetable, or enhancing the longevity of fruit or vegetableripeness. The method includes providing a transgenic plant transformedwith a DNA construct including a first nucleic acid molecule encoding apolypeptide according to the first or second aspect of the invention ora fusion protein according to the third aspect of the invention; andgrowing the plant under conditions effective to permit the DNA constructto express the peptide or the fusion polypeptide to impart desiccationresistance to cuttings removed from a transgenic ornamental plant,impart post-harvest disease resistance or desiccation resistance to afruit or vegetable removed from the transgenic plant, or enhancelongevity of ripeness for a fruit or vegetable removed from thetransgenic plant.

A sixteenth aspect of the invention relates to a method of impartingdisease resistance to plants, enhancing plant growth, impartingtolerance and resistance to biotic stressors, imparting tolerance toabiotic stress, or modulating biochemical signaling. This methodincludes providing a transgenic plant seed transformed with a DNAconstruct according to the thirteenth aspect of the invention; plantingthe transgenic plant seed in soil; and propagating a transgenic plantfrom the transgenic plant seed to permit the DNA construct to expressthe peptide or the fusion polypeptide to impart disease resistance,enhance plant growth, impart tolerance to biotic stress, or imparttolerance to abiotic stress to the transgenic plant.

A seventeenth aspect of the invention relates to a method of impartingdesiccation resistance to cuttings removed from ornamental plants,imparting post-harvest disease or post-harvest desiccation resistance toa fruit or vegetable, or enhancing the longevity of fruit or vegetableripeness. The method includes providing a transgenic plant seedtransformed with a DNA construct according to the thirteenth aspect ofthe invention; planting the transgenic plant seed in soil; andpropagating a transgenic plant from the transgenic plant seed to permitthe DNA construct to express the peptide or the fusion polypeptide toimpart desiccation resistance to cuttings removed from a transgenicornamental plant, impart post-harvest disease resistance or desiccationresistance to a fruit or vegetable removed from the transgenic plant, orenhance longevity of ripeness for a fruit or vegetable removed from thetransgenic plant.

By providing HR-eliciting peptides and active but non-HR-elicitingpeptides that exhibit improved solubility, stability, resistance tochemical degradation, or a combination of these properties, it willafford growers with greater flexibility in preparing, handling, anddelivering to plants in their fields or greenhouses effective amounts ofcompositions containing these HR-eliciting and non-HR-elicitingpeptides. Simplifying the application process for growers will lead togreater compliance and, thus, improved results with respect to one ormore of disease resistance, growth enhancement, tolerance and resistanceto biotic stressors, tolerance to abiotic stress, desiccation resistancefor cuttings removed from ornamental plants, post-harvest diseaseresistance or desiccation resistance to fruit or vegetables harvestedfrom plants, and/or improved longevity of fruit or vegetable ripenessfor fruit or vegetables harvested from plants. These and other benefitsare described herein, and the utility of these peptides is demonstratedby the accompanying Examples where resistance to tobacco mosaic viruswas shown in tobacco, resistance to nematodes was demonstrated in soyand tomato, and drought resistance was demonstrated in soy and corn.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a solubility and stability test of peptide P5 (SEQ ID NO:8) in the following 50 mM buffer solutions: citrate, pH 7.2; EDDS, pH7.3; imidazole, pH 7.5; EDTA, pH 8.0; sodium phosphate, pH 8.0; and TES,pH 8.0. Peptide P5 exhibited poor solubility below pH 7.0, and exhibitedits best results in an EDTA buffer at pH 8.0 (about 40% remaining after14 days).

FIG. 2 shows a solubility and stability test of peptide P5-9 (SEQ ID NO:15) in deionized water and the following 50 mM buffer solutions:citrate, pH 5.6; MES, pH 6.0; MOPS, pH 6.5; citrate, pH 7.2; EDDS, pH7.3; imidazole, pH 7.5; EDTA, pH 8; sodium phosphate, pH 8.0; and TES,pH 8.0. Peptide P5-9 exhibited better solubility performance at 500ug/ml as well as better stability compared with P5. In both citrate atpH 7.2 and phosphate at pH 8.0, peptide P5-9 exhibited around 80%remaining after 45 days.

FIG. 3 shows a solubility and stability test of peptide P5-21 (SEQ IDNO: 33) in the following 50 mM buffer solutions: citrate, pH 5.6; MES,pH 6.0; MOPS, pH 6.5; citrate, pH 7.2; EDDS, pH 7.3; imidazole, pH 7.5;EDTA, pH 8; sodium phosphate, pH 8.0; and TES, pH 8.0. Peptide P5-21exhibited good solubility at pH 5.6 and higher, as well as excellentstability (>90% after 14 days) for all buffers pH >7.0 except EDTA(around 80%).

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to novel peptides that possess theability to either induce a hypersensitive response in plants or promoteactive plant responses (or both) that afford one or more of thefollowing attributes: disease resistance, growth enhancement, toleranceand resistance to biotic stressors, tolerance to abiotic stress,desiccation resistance for cuttings removed from ornamental plants,post-harvest disease resistance or desiccation resistance to fruit orvegetables harvested from plants, and/or improved longevity of fruit orvegetable ripeness for fruit or vegetables harvested from plants. Theinduction of these plant responses involves modulating plant biochemicalsignaling.

As used herein, naturally occurring amino acids are identifiedthroughout by the conventional three-letter and/or one-letterabbreviations, corresponding to the trivial name of the amino acid, inaccordance with the following list: Alanine (Ala, A), Arginine (Arg, R),Asparagine (Asn, N), Aspartic acid (Asp, D), Cysteine (Cys, C), Glutamicacid (Glu, E), Glutamine (Gln, Q), Glycine (Gly, G), Histidine (His, H),Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met,M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine(Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).The abbreviations are accepted in the peptide art and are recommended bythe IUPAC-IUB commission in biochemical nomenclature. Naturallyoccurring variations of these amino acids are well known and include,without limitation, gamma-glutamate (g-Glu) and isoaspartate (iso-Asp orisoD).

The term “amino acid” further includes analogues, derivatives, andcongeners of any specific amino acid referred to herein, as well asC-terminal or N-terminal protected amino acid derivatives (e.g.,modified with an N-terminal, C-terminal, or side-chain protecting group,including but not limited to acetylation, formylation, methylation,amidation, esterification, PEGylation, and addition of lipids.Non-naturally occurring amino acids are well known and can be introducedinto peptides of the present invention using solid phase synthesis asdescribed below. Furthermore, the term “amino acid” includes both D- andL-amino acids. Hence, an amino acid which is identified herein by itsname, three letter or one letter symbol and is not identifiedspecifically as having the D or L configuration, is understood to assumeany one of the D or L configurations. In one embodiment, a peptidecomprises all L-amino acids.

In certain embodiments, peptides are identified to “consist of” arecited sequence, in which case the peptide includes only the recitedamino acid sequence(s) without any extraneous amino acids at the N- orC-terminal ends thereof. To the extent that a recited sequence is in theform of a consensus sequence where one or more of the denoted X or Xaaresidues can be any of one or more amino acids, then multiple peptidesequences are embraced by a peptide consisting of such a recitedsequence.

In certain other embodiments, peptides are identified to “consistessentially of” a recited sequence, in which case the peptide includesthe recited amino acid sequence(s) optionally with one or moreextraneous amino acids at the N- and/or C-terminal ends thereof, whichextraneous amino acids do not materially alter one or more of thefollowing properties: (i) the ability of the peptide to induce ahypersensitive response or other active response in plants, (ii)solubility of the peptide in water or aqueous solutions, (iii) stabilityof the peptide dissolved in water or aqueous solution at 50° C. over aperiod of time (e.g., 3 weeks), and (iv) resistance of the peptide tochemical degradation in the presence of an aqueous buffered solutionthat includes a biocidal agent (e.g., Proxel®GXL) at 50° C. over aperiod of time (e.g., 3 weeks).

Briefly, the stability and resistance to chemical degradation ofpeptides can be assessed as follows using peptide samples having aninitial purity of at least about 80%, at least about 82%, at least about84%, at least about 86%, at least about 88%, at least about 90%, atleast about 92%, at least about 94%, at least about 96%, or at leastabout 98%. For water stability, the peptide is dissolved directly inde-ionized water. For chemical degradation tests, the peptide isdissolved in an aqueous solution containing 50 mM pH buffer and 0.25%Proxel GXL. Exemplary pH buffers include, without limitation: (i)Citrate pH 5.6; (ii) MES pH 6.0; (iii) MOPS pH 6.5; (iv) imidazole pH7.5; (v) Citrate pH 7.2; (vi) EDDS, pH 7.3; (vii) EDTA pH 8.0; (viii)sodium phosphate pH 8.0; or (ix) TES pH 8.0. Peptides are firstdissolved in the aqueous solution at a concentration of 0.5 mg/ml. Thesamples are incubated at 50° C. to allow for accelerated degradation. Aninitial sample of the peptide is removed, diluted 10× with water, andanalyzed by reverse-phase HPLC. Briefly, 20 μl of the sample is injectedinto the solvent flow of an HPLC instrument and analyzed on a C18 HPLCcolumn (YMC ProPack C18, YMC, Japan, or C18 Stablebond, AgilentTechnologies, USA) using either a triethylamine phosphate inwater/acetonitrile gradient or a 0.1% TFA in water/0.1% TFA inacetonitrile gradient to separate different peptide species. Elutingpeptides are monitored by UV absorbance at 218 nm and quantified basedon the area under the peak. The area under the peak for the initialpeptide sample is treated as the standard for relative quantification insubsequent runs. At regular intervals (e.g., 1, 3, 7, 10, and 14 days),each peptide sample is surveyed and analyzed by HPLC as described above.If necessary to observe degradation (i.e., where the peptide exhibits ahigh degree of chemical stability), this protocol can be extended byseveral weeks to observe degradation. The quantification of subsequentpeptide runs is expressed as a percentage of the original (day 0) HPLCresult.

A peptide that is at least partially soluble in water or aqueoussolution exhibits a solubility of greater than 0.1 mg/ml, preferably atleast about 1.0 mg/ml, at least about 2.0 mg/ml, at least about 3.0mg/ml, or at least about 4.0 mg/ml. In certain embodiments, the peptideexhibits high solubility in water or aqueous solution, with a solubilityof at least about 5.0 mg/ml, at least about 10.0 mg/ml, at least about15.0 mg/ml, or at least about 20 mg/ml.

A peptide that is stable in water or aqueous solution exhibits at leastabout 66%, at least about 68%, at least about 70%, at least about 72%,at least about 74%, at least about 76%, at least about 78%, at leastabout 80%, at least about 82%, at least about 84%, at least about 86%,at least about 88%, or at least about 90% of the original peptideconcentration over the designated period of time incubated at 50° C. Incertain embodiments, the designated period of time is 3 days, 7 days, 14days, 21 days, 28 days, one month, two months, three months, or fourmonths.

A peptide that is resistant to chemical degradation exhibits at leastabout 66%, at least about 68%, at least about 70%, at least about 72%,at least about 74%, at least about 76%, at least about 78%, at leastabout 80%, at least about 82%, at least about 84%, at least about 86%,at least about 88%, or at least about 90% of the original peptideconcentration over the designated period of time incubated at 50° C. Incertain embodiments, the designated period of time is 3 days, 7 days, 14days, 21 days, 28 days, one month, two months, three months, or fourmonths.

A property of a peptide to elicit a hypersensitive response, or not,upon infiltration or application of the peptide to plant tissues can bemeasured by applying the peptide in dry powder form or in solution formto a plant, particularly though not exclusively a plant leaf.Application rates include 1-500 ug/ml for liquid solution and0.0001-0.5% (w/w for powder application. Exemplary application of thepeptide in solution form is described in the accompanying Examples.Plants are considered HR-positive (“HR+”) if they exhibit wide-spreadmacroscopic cell death visible to the naked eye, accompanied by wiltingand browning of the affected tissue within 48 hours. Plants areconsidered HR-negative (“HR−”) if they exhibit no discernible wilting ortissue death observable by naked eye.

In certain embodiments, material alteration of the one or moreproperties is intended to mean that there is less than 20% variation,less than 15% variation, less than 10% variation, or less than 5%variation in a recited property when comparing a peptide possessing theone or more extraneous amino acids to an otherwise identical peptidelacking the one or more extraneous amino acids. In certain embodiments,the number of extraneous amino acids at the N- or C-terminal ends is upto 20 amino acids at one or both ends, up to 15 amino acids at one orboth ends, up to 10 amino acids at one or both ends, up to 7 amino acidsat one or both ends, up to 5 amino acids at one or both ends, or up to 3amino acids at one or both ends. Further, to the extent that a recitedsequence is in the form of a consensus sequence where one or more of thedenoted X or Xaa residues can be any of one or more amino acids, thenmultiple peptide sequences are embraced by the peptide consistingessentially of such a recited sequence, without regard to additionalvariations of such sequences that are afforded by the presence ofextraneous amino acids at the N- and/or C-terminal ends thereof.

In various embodiments of the invention, the disclosed peptides mayinclude a hydrophilic amino acid sequence, e.g., at either theN-terminal or C-terminal end of a designated peptide sequence. Thehydrophilic amino acid sequence is at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, or at least 10 aminoacids in length, and includes amino acid residues that contribute to ahydrophilic property of the amino acid sequence that is adjacent to theamino acid sequence of the designated peptide (i.e., the peptide thatinduces an active plant response). Different methods have been used inthe art to calculate the relative hydrophobicity/hydrophilicity of aminoacid residues and proteins (Kyte et al., “A Simple Method for Displayingthe Hydropathic Character of a Protein,” J. Mol. Biol. 157: 105-32(1982); Eisenberg D, “Three-dimensional Structure of Membrane andSurface Proteins,” Ann. Rev. Biochem. 53: 595-623 (1984); Rose et al.,“Hydrogen Bonding, Hydrophobicity, Packing, and Protein Folding,” Annu.Rev. Biomol. Struct. 22: 381-415 (1993); Kauzmann, “Some Factors in theInterpretation of Protein Denaturation,” Adv. Protein Chem. 14: 1-63(1959), which are hereby incorporated by reference in their entirety).Any one of these hydrophobicity scales can be used for the purposes ofthe present invention; however, the Kyte-Doolittle hydrophobicity scaleis perhaps the most often referenced scale. These hydropathy scalesprovide a ranking list for the relative hydrophobicity of amino acidresidues. For example, amino acids that contribute to hydrophilicityinclude Arg (R), Lys (K), Asp (D), Glu (E), Gln (Q), Asn (N), and His(H) as well as, albeit to a lesser extent, Ser (S), Thr (T), Gly (G),Pro (P), Tyr (Y), and Trp (W). For example, polyglutamate sequences canbe used to enhance solubility of proteins and other drug molecules(Lilie et al, Biological Chemistry 394(8):995-1004(2013); Li et al.,Cancer Research 58: 2404-2409(1998)), each of which is herebyincorporated by reference in its entirety).

The “hydropathy index” of a protein or amino acid sequence is a numberrepresenting its average hydrophilic or hydrophobic properties. Anegative hydropathy index defines the hydrophilicity of the amino acidsequence of interest. The hydropathy index is directly proportional tothe hydrophilicity of the amino acid sequence of interest; thus, themore negative the index, the greater its hydrophilicity. In certainembodiments, the added hydrophilic amino acid sequence described abovehas a hydropathy index of less than 0, −0.4, −0.9, −1.3, −1.6, −3.5,−3.9, or −4.5. In certain embodiments, the resulting entire peptide willhave a hydropathy index of less than 0.7, 0.3, 0.2, 0.1, or 0.0,preferably less than −0.1, −0.2, −0.3, −0.4, more preferably less than−0.5, −0.6, −0.7, −0.8, −0.9, or −1.0.

In the peptides of the present invention, amino acids that contribute toa hydrophilic hydropathy index, for either the peptide as a whole or theadded hydrophilic amino acid sequence, include Arg (R), Lys (K), Asp(D), Glu (E), Gln (Q), Asn (N), His (H), Ser (S), Thr (T), Gly (G), Pro(P), Tyr (Y), and Trp (W). Of these, Asp (D), Glu (E), Gln (Q), Asn (N)or their variants are preferred. Exemplary variants include g-glutamatefor Glu and isoaspartic acid (or isoD) for Asp.

As used herein, in this and in other aspects of the invention, the term“hydrophobic amino acid” is intended to refer to an amino acid thatcontributes hydrophobicity to the hydropathy index of a designated aminoacid sequence. Amino acids that contribute to a hydrophobic hydropathyindex, for either the peptide as a whole or a particular amino acidsequence thereof, include Ile (I), Val (V), Leu (L), Phe (F), Cys (C),Met (M), and Ala (A). In certain embodiments, the term “hydrophobicamino acid” may refer to any one of Ile (I), Val (V), Leu (L), Phe (F),Cys (C), Met (M), and Ala (A); or, alternatively, to any one of Ile (I),Val (V), Leu (L), Phe (F), and Ala (A). In certain other embodiments,the term “hydrophobic amino acid” may refer to one of Ile (I), Val (V),Leu (L), and Phe (F).

As used herein, the term “non-hydrophobic amino acid” is intended tomean an amino acid that is hydrophilic (or not hydrophobic) on one ofthe above-identified hydrophobicity scales. This term generally refersto those amino acids that contribute to a hydrophilic hydropathy indexfor either the peptide as a whole or the added hydrophilic amino acidsequence.

In one aspect of the invention, the peptide includes the amino acidsequence of:

(i)  (SEQ ID NO: 1) (L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X-(L/I/V/F),  or (ii)  (SEQ ID NO: 2)L-X-X-L-L-L-X-(F/L)-(I/L)-X-X-X-L,wherein X for both SEQ ID NO: 1 and 2 at position 3 is optional and,when present, is any amino acid; and each X at positions 2, 7, 10, 11,and 12 is any amino acid.

In a related aspect of the invention, the peptide includes the aminoacid sequence of SEQ ID NO: 1 or 2 (shown above), wherein the peptide isfree of cysteine or free of both cysteine and methionine; X at position3 is optional and, when present, is any amino acid; and each X atpositions 2, 7, 10, 11, and 12 is any amino acid.

According to one embodiment, X at position 3 is not present (i.e., thegap between the first hydrophobic amino acid and the first hydrophobicamino acid triplet is reduced from two to one amino acid residue. Inthis embodiment, it is contemplated that these peptides exclude theamino acid at only position 3.

In an alternative embodiment, X at position 3 is present (i.e., the gapbetween the hydrophobic amino acids is maintained at two amino acidresidues).

The peptide length in this embodiment is less than 100 amino acids, oralternatively less than 90 amino acids, less than 80 amino acids, lessthan 70 amino acids, less than 60 amino acids, or less than about 50amino acids. In certain embodiments, the peptide length is between 12and about 50 amino acids in length.

In the embodiments described above, where X at each of positions 2, 3(when present), 7, 10, 11, and 12 of SEQ ID NO: 1 and 2 can be any aminoacid, in certain embodiments these residues are hydrophilic in nature.As described above, these hydrophilic amino acids include Arg (R), Lys(K), Asp (D), Glu (E), Gln (Q), Asn (N), His (H), Ser (S), Thr (T), Gly(G), Pro (P), Tyr (Y), and Trp (W). Of these, Asp (D), Glu (E), Gln (Q),Asn (N) or their variants are preferred. Exemplary variants includeg-glutamate for Glu and isoaspartic acid (or isoD) for Asp.

In certain embodiments, X at positions 2 and 3 (when present) isselected independently from Asp (D), Glu (E), and Gln (Q).

In certain embodiments, X at positions 7, 10, 11, and 12 is selectedindependently from Ala (A), Met (M), Gly (G), Ser (S), and Glu (E).

In this embodiment, the isolated peptide is stable when dissolved inwater; resistant to chemical degradation in aqueous conditions in thepresence of a pH buffer and a biocide, as described above; and/or has asolubility in an aqueous solution of at least about 1.0 mg/ml.

One set of peptides according to the first aspect of the invention havethe amino acid sequence of:

(SEQ ID NO: 3) (Q/E)-(Q/E)-(L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X-(L/I/V/F)- (D/G/Q/E)-(D/G/Q/E),,wherein X at position 5 is optional and, when present, is any aminoacid; and each X at positions 4, 9, 12, 13, and 14 is any amino acid. Inone embodiment, X at position 5 is present. In another embodiment, X atposition 5 is not present.

In the embodiments described above, where X at each of positions 4, 5(when present), 9, 12, 13, and 14 of SEQ ID NO: 3 can be any amino acid,in certain embodiments these residues are hydrophilic in nature. Asdescribed above, these hydrophilic amino acids include Arg (R), Lys (K),Asp (D), Glu (E), Gln (Q), Asn (N), His (H), Ser (S), Thr (T), Gly (G),Pro (P), Tyr (Y), and Trp (W). Of these, Asp (D), Glu (E), Gln (Q), Asn(N) or their variants are preferred. Exemplary variants includeg-glutamate for Glu and isoaspartic acid (or isoD) for Asp.

In certain embodiments, X at positions 4 and 5 (when present) isselected independently from Asp (D), Glu (E), and Gln (Q).

In certain embodiments, X at positions 9 and 12 to 14 is selectedindependently from Ala (A), Met (M), Gly (G), Ser (S), and Glu (E).

In certain embodiments, these peptides also meet the structural featuresdefining the peptides of SEQ ID NO: 1, in which case methionine andcysteine residues are not present.

Another set of peptides according to the first aspect of the inventionhave the amino acid of:

(SEQ ID NO: 4) (L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X-(L/I/V/F)-(D/G/Q/E)- (D/G/Q/E),,wherein X at position 3 is optional and, when present, is any aminoacid; and each X at positions 2, 7, 10, 11, and 12 is any amino acid. Inone embodiment, X at position 3 is present. In another embodiment, X atposition 3 is not present.

In the embodiments described above, where X at each of positions 2, 3(when present), 7, 10, 11, and 12 of SEQ ID NO: 4 can be any amino acid,in certain embodiments these residues are hydrophilic in nature. Asdescribed above, these hydrophilic amino acids include Arg (R), Lys (K),Asp (D), Glu (E), Gln (Q), Asn (N), His (H), Ser (S), Thr (T), Gly (G),Pro (P), Tyr (Y), and Trp (W). Of these, Asp (D), Glu (E), Gln (Q), Asn(N) or their variants are preferred. Exemplary variants includeg-glutamate for Glu and isoaspartic acid (or isoD) for Asp.

In certain embodiments, X at positions 2 and 3 (when present) isselected independently from Asp (D), Glu (E), and Gln (Q).

In certain embodiments, X at positions 7, 10, 11, and 12 is selectedindependently from Ala (A), Met (M), Gly (G), Ser (S), and Glu (E).

A further set of peptides according to the first aspect of the inventionhave the amino acid sequence of:

(SEQ ID NO: 5) (Q/E)-(Q/E)-(L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X-(L/I/V/F),,wherein X at position 5 is optional and, when present, is any aminoacid; and each X at positions 4, 9, 12, 13, and 14 is any amino acid. Inone embodiment, X at position 5 is present. In another embodiment, X atposition 5 is not present.

In the embodiments described above, where X at each of positions 4, 5(when present), 9, 12, 13, and 14 of SEQ ID NO: 3 can be any amino acid,in certain embodiments these residues are hydrophilic in nature. Asdescribed above, these hydrophilic amino acids include Arg (R), Lys (K),Asp (D), Glu (E), Gln (Q), Asn (N), His (H), Ser (S), Thr (T), Gly (G),Pro (P), Tyr (Y), and Trp (W). Of these, Asp (D), Glu (E), Gln (Q), Asn(N) or their variants are preferred. Exemplary variants includeg-glutamate for Glu and isoaspartic acid (or isoD) for Asp.

In certain embodiments, X at positions 4 and 5 (when present) isselected independently from Asp (D), Glu (E), and Gln (Q).

In certain embodiments, X at positions 9 and 12 to 14 is selectedindependently from Ala (A), Met (M), Gly (G), Ser (S), and Glu (E).

Another aspect of the invention relates to an isolated peptidecomprising the amino acid sequence of

(SEQ ID NO: 6) J-X-X-J-J-J-X-J-J-X-X-X-Jwherein X at position 3 is optional and, when present, is any aminoacid; and each X at positions 2, 7, 10, 11, and 12 is any amino acid;one to three of the J residues at positions 1, 4, 5, 6, 8, 9, and 13 isa non-hydrophobic amino acid or A, and all other of the J residues areL, I, V, or F, and the peptide induces an active plant response, butdoes not induce a hypersensitive response, when applied to plant tissue.These active plant responses afford one or more of the followingattributes: modified biochemical signaling; enhanced growth; pathogenresistance; and/or biotic or abiotic stress resistance.

In the embodiments described above, where X at each of positions 2, 3(when present), 7, 10, 11, and 12 of SEQ ID NO: 6 can be any amino acid,in certain embodiments these residues are hydrophilic in nature. Asdescribed above, these hydrophilic amino acids include Arg (R), Lys (K),Asp (D), Glu (E), Gln (Q), Asn (N), His (H), Ser (S), Thr (T), Gly (G),Pro (P), Tyr (Y), and Trp (W). Of these, Asp (D), Glu (E), Gln (Q), Asn(N) or their variants are preferred. Exemplary variants includeg-glutamate for Glu and isoaspartic acid (or isoD) for Asp.

In certain embodiments, X at positions 2 and 3 (when present) isselected independently from Asp (D), Glu (E), and Gln (Q).

In certain embodiments, X at positions 7, 10, 11, and 12 is selectedindependently from Ala (A), Met (M), Gly (G), Ser (S), and Glu (E).

In this embodiment, the isolated peptide is stable when dissolved inwater; resistant to chemical degradation in aqueous conditions in thepresence of a pH buffer and a biocide, as described above; and/or has asolubility in an aqueous solution of at least about 1.0 mg/ml.

Exemplary peptides that meet the consensus structure of one or more ofSEQ ID NO: 1 to 6 are identified in Table 1 and Table 2 below:

TABLE 1 Peptide Variants of Peptide P5/P5A (SEQ ID NOS: 8 and 9) SEQPeptide Name Sequence ID NO: wildtype SAGSEQQLDQLLAMFIMMMLQQ   7 P5SAGSEQQLDLLLMFIMMMLQQ   8 P5A SAGSEQQLDQLLLMFIMMMLQQ   9 P5-2SAGSEQQLDLLLMFIAAALQQ  10 P5-3 SAGSEQQLDLLLAFIAAALQQ  11 P5-4SAGSEQQLELLLAFIAAALQQ  12 P5-7 SEEEEELDLLLAFIAAAL  13 P5-8SEEEEELDLLLAFIAAALQQ  14 P5-9 LDLLLAFIAAALEEEEEE  15 P5-10LDLLLAFIEEELEEEE  16 P5-11 SEEELDLLLAFIAAALEE  17 P5-12SEEELDLLLAFIEEELEE  18 P5-13 SEEELDLLLAFIAAALDD  19 P5-14SEEEEELDLLLAFIAAALGG  20 P5-1000 SEEEEEEELDLLLAFIAAALAA  21 P5-1001SEEEEEELDLLLAFIAAALSS  22 P5-1002 SAGSEQQLDLLLGFIGGGLQQ  23 P5-1003SAGSEQQLDLLLSFISSSLQQ  24 P5-15 SEEEEELDLLLAFIAAALQ  25 P5-16SEEEEELDLLLAFIAAALS  26 P5-17 SEEEEELDLLLAFIAAALA  27 P5-18SEEEEELDLLLAFIAAALE  28 P5-19 SELELLLAFIAAALEEEEE  29 P5-22SEEELELLLAFIAAALEEEE  30 P5-1004 SELDLLLAFIAAALEEEEE  31 P5-1005SEEEEELDLLLALIAAALQQ  32 P5-21 SEEQLELLLAFIAAALQQEE  33 P5-26SEEQLDLLLAFIAAALQEE  34 P5-1006 SELELLLAFIEEELEE  35 P5-20SELELLLEFIEEELEE  36 P5-1007 SELELLLEFIEEELE  37 P5-1008 SELELLLELIEEELE 38 P5-48 SEEQLDLLLEFIEEELQQEE  39 P5-1009 SELDLLLAFIDDDLEEEEE  40 P5-29SEEELDLLLMFIMMMLEE  42 P5-31 SEEEQLDLLLMFIMMMLEE  43 P5-33SEEEQLDLLLMFIMMMLQEE  44 P5-47 SEEQLDLLLMFIMMMLQQEE  45 P5-1010LELLLEFIEEELE  46 P5-1011 LELLLEFIEEELEE  47 P5-1012 SELELLLEFIEEEL  48P5A variant LDQLLLMFIMMMLQQ  49 P5-23 SEEEEELDQLLLAFIAAALQQ  50 P5-24SEEEEELDQLLLAFIAAAL  51 P5-25 SEEEEQLDQLLLAFIAAALQQ  52 P5-27SEEQLDQLLLAFIAAALQEE  53 P5-28 SEEQLDQLLLAFIAAALEE  54 P5-30SEEELDQLLLMFIMMMLEE  55 P5-32 SEEEQLDQLLLMFIMMMLEE  56 P5-34SEEEQLDQLLLMFIMMMLQEE  57 P5-46 SEEQLDQLLLMFIMMMLQQEE  58P5A variant M to A LDQLLLAFIAAALQQ  59 P5A variant M to ELDQLLLEFIEEELQQ  60 P5A variant QLDQLLLMFIMMMLQQ  61 P5A variant M to AQLDQLLLAFIAAALQQ  62 P5A variant M to E QLDQLLLEFIEEELQQ  63 P5A variantQQLDQLLLMFIMMMLQQ  64 P5A variant M to A QQLDQLLLAFIAAALQQ  65P5A variant M to E QQLDQLLLEFIEEELQQ  66 P5A variant LDQLLLMFIMMML  67P5A variant M to A LDQLLLAFIAAAL  68 P5A variant M to E LDQLLLEFIEEEL 69 P5A variant Q to E SAGSEEELDQLLLMFIMMMLEE  70 P5 variant Q to ESAGSEEELDLLLMFIMMMLEE  41 P5 mut 8E P5-35 SAGSEQQEDLLLMFIMMMLQQ  71P5 mut 10E P5-36 SAGSEQQLDELLMFIMMMLQQ  72 P5 mut 11E P5-37SAGSEQQLDLELMFIMMMLQQ  73 P5 mut 12E P5-38 SAGSEQQLDLLEMFIMMMLQQ  74P5 mut 13E P5-39 SAGSEQQLDLLLEFIMMMLQQ  75 P5 mut 14E P5-40SAGSEQQLDLLLMEIMMMLQQ  76 P5 mut 15E P5-41 SAGSEQQLDLLLMFEMMMLQQ  77P5 mut 16E P5-42 SAGSEQQLDLLLMFIEMMLQQ  78 P5 mut 17E P5-43SAGSEQQLDLLLMFIMEMLQQ  79 P5 mut 18E P5-44 SAGSEQQLDLLLMFIMMELQQ  80P5 mut 19E P5-45 SAGSEQQLDLLLMFIMMMEQQ  81 P5 mut 9KSAGSEQQLKLLLMFIMMMLQQ  82 P5 SS mut SAGSESSLDLLLMFIMMMLQQ  83 P5 SS mutSAGSEQQLDLLLMFIMMMLSS  84 P5 GG mut SAGSEGGLDLLLMFIMMMLQQ  85 P5 GG mutSAGSEQQLDLLLMFIMMMLGG  86 P5 single mut 8V SAGSEQQVDLLLMFIMMMLQQ  87P5 single mut 10V SAGSEQQLDVLLMFIMMMLQQ  88 P5 single mut 11VSAGSEQQLDLVLMFIMMMLQQ  89 P5 single mut 12V SAGSEQQLDLLVMFIMMMLQQ  90P5 single mut 13V SAGSEQQLDLLLVFIMMMLQQ  91 P5 single mut 14VSAGSEQQLDLLLMVIMMMLQQ  92 P5 single mut 15V SAGSEQQLDLLLMFVMMMLQQ  93P5 single mut 16V SAGSEQQLDLLLMFIVMMLQQ  94 P5 single mut 17VSAGSEQQLDLLLMFIMVMLQQ  95 P5 single mut 18V SAGSEQQLDLLLMFIMMVLQQ  96P5 single mut 19V SAGSEQQLDLLLMFIMMMVQQ  97 P5A single mut 8E, P5-53SAGSEQQEDQLLLMFIMMMLQQ  98 P5A single mut 11E, P5-54SAGSEQQLDQELLMFIMMMLQQ  99 P5A single mut 12E SAGSEQQLDQLELMFIMMMLQQ 100P5A single mut 13E SAGSEQQLDQLLEMFIMMMLQQ 101 P5A single mut 14ESAGSEQQLDQLLLEFIMMMLQQ 102 P5A single mut 15E SAGSEQQLDQLLLMEIMMMLQQ 103P5A single mut 16E SAGSEQQLDQLLLMFEMMMLQQ 104 P5A single mut 17ESAGSEQQLDQLLLMFIEMMLQQ 105 P5A single mut 18E SAGSEQQLDQLLLMFIMEMLQQ 106P5A single mut 19E SAGSEQQLDQLLLMFIMMELQQ 107 P5A single mut 20ESAGSEQQLDQLLLMFIMMMEQQ 108 P5A single mut 9K SAGSEQQLKQLLLMFIMMMLQQ 109P5A single mut 10E SAGSEQQLDELLLMFIMMMLQQ 110 P5A single mut 10SSAGSEQQLDSLLLMFIMMMLQQ 111 P5A single mut 10A SAGSEQQLDALLLMFIMMMLQQ 112P5A SS mut SAGSESSLDQLLLMFIMMMLQQ 113 P5A SS mut SAGSEQQLDQLLLMFIMMMLSS114 P5A GG mut SAGSEGGLDQLLLMFIMMMLQQ 115 P5A GG mutSAGSEQQLDQLLLMFIMMMLGG 116 P5A single mut 8V SAGSEQQVDQLLLMFIMMMLQQ 117P5A single mut 11V SAGSEQQLDQVLLMFIMMMLQQ 118 P5A single mut 12VSAGSEQQLDQLVLMFIMMMLQQ 119 P5A single mut 13V SAGSEQQLDQLLVMFIMMMLQQ 120P5A single mut 14V SAGSEQQLDQLLLVFIMMMLQQ 121 P5A single mut 15VSAGSEQQLDQLLLMVIMMMLQQ 122 P5A single mut 16V SAGSEQQLDQLLLMFVMMMLQQ 123P5A single mut 17V SAGSEQQLDQLLLMFIVMMLQQ 124 P5A single mut 18VSAGSEQQLDQLLLMFIMVMLQQ 125 P5A single mut 19V SAGSEQQLDQLLLMFIMMVLQQ 126P5A single mut 20V SAGSEQQLDQLLLMFIMMMVQQ 127 P5-1013SAGSEQQLDQLLLMFIMMML 128 P5-1014 QQLDQLLLMFIMMML 129 P5-55SAGSEQQLDQLLLAFIAAALQQ 130 P5-1015 SAGSEQQLDQLLLEFIEEELQQ 131 P5-1016QQLDLLLMFIMMMLQQ 132 P5-1017 LDLLLMFIMMMLQQ 133 P5-1018SAGSEQQLDLLLMFIMMML 134 P5-1019 QQLDLLLMFIIMMML 135 P5-1020 LDLLLMFIMMML136 P5-1021 SAGSEQQLDLLLEFIEEELQQ 137 Cleavable tag seqHHHHHHRQQLDLLLAFIAAALQQ 138 P5-5 QLELLLAFIAAALQQ 139 P5-6SAGSEQQLDLLLAFIAAAL 140 P5-49 SAGSEQQEDLLLAFIAAALQQ 510 P5-50SAGSEQQLDELLAFIAAALQQ 511 P5-51 SAGSEQQEDELLAFIAAALQQ 512 P5-52SAGSEQQEDELLMFIMMMLQQ 513 P5-56 SEEQEELLLAFIAAALQQEE 514 P5-57SEEQLEELLAFIAAALQQEE 515 P5-58 SEEQEEELLAFIAAALQQEE 516 P5-59, P5-RSAGSEQQLDLLLMFIMMMLQQR 517 P5-60, P5-21-R SEEQLELLLAFIAAALQQEER 518P5-61 SAGSEQQEDLLLAFIALQQ 519 P5-62 SAGSEQQLDELLAFIALQQ 520 P5-63SAGSEQQLDLLLEFIALQQ 521 P5-64 SAGSEQQLDLLLAEIALQQ 522 P5-65SAGSEQQLDLLLAFIEALQQ 523 P5-66 SAGSEQQLDLLLAFIAEALQQ 524 P5-67SAGSEQQLDLLLAFIEAALQQ 525 P5-68 SAGSEQQLDLLLAFIAAELQQ 526 P5-69SAGSEQQLDLLLAEIAAALQQ 527 P5-70 SAGSEQQLDLLLEFIAAALQQ 528 P5-71SAGSEQQEDLLLAFIAAALQQ 529 P5-72 SAGSEQQLDELLAFIAAALQQ 530 P5-73SQAGSEQLDLLLMFIMMMLQQ 531 P5-74 AEQGSSQLDLLLMFIMMMLQQ 532 P5-75NQGISEKQQLDLLLMFIMMMLQQ 533 P5-76 NQGISEKQQLDLLLAFIAAALQQ 534 P5-77NFGTPDSTVQNPQDASKPNQLDLLLMFIMMMLQQ 535 P5-78NFGTPDSTVQNPQDASKPNQLDLLLAFIAAALQQ 536 P5-79ITPDGQGGGQIGDNPQLDLLLMFIMITMLQQ 537 P5-80 ITPDGQGGGQIGDNPQLDLLLAFIAAALQQ538 P5-87 SEEQLDLLLAFIAAALQQEE 539 P5-88 SEEQLELLLAFIAAALQEE 540 P5-89SAGSEEELDLLLMFIMMMLQQ 541 P5-90 SAGSEQQLDLLLMFIMMMLEE 542 P5-91SEEQQLDLLLMFIMMMLQQEE 543 P5-92 SEEEEQLDQLLLAFIAAALQQR 544 P5-1022QLEQLLAFIAAALQQ 545 P5-1023 QLDLLLAFIAAALQQ 546Select peptides in Table 1 include N- or C-terminal solubility tags,indicated by italic print, including SEEEEEEE, SEEEEEE, SEEEEE, SEEEE,SEEE, SEE, EE, EE, and EEE. Peptides comprising the sequences shown inTable 1 but lacking these specific solubility tags (or having adifferent solubility tag) are also contemplated herein.

TABLE 2 Peptide Variants of Peptide P5/P5A (SEQ ID NOS: 8 and 9) SEQP5/P5A Core/Core Variants ID NO: LDLLLMFIMMML 136 LDQLLLMFIMMML  67LDLLL(A/E)FIMMML 141 LDLLLMFI(A/E)MML 142 LDLLLMFIM(A/E)ML 143LDLLLMFIMM(A/E)L 144 LDLLL(A/E)FI(A/E)MML 145 LDLLL(A/E)FIM(A/E)ML 146LDLLL(A/E)FIMM(A/E)L 147 LDLLLMFI(A/E)(A/E)ML 148 LDLLLMFI(A/E)M(A/E)L149 LDLLLMFIM(A/E)(A/E)L 150 LDLLLMFI(A/E)(A/E)(A/E)L 151LDLLL(A/E)FIM(A/E)(A/E)L 152 LDLLL(A/E)FI(A/E)M(A/E)L 153LDLLL(A/E)FI(A/E)(A/E)ML 154 LDLLL(A/E)FI(A/E)(A/E)(A/E)L 155LDQLLL(A/E)FIMMML 156 LDQLLLMFI(A/E)MML 157 LDQLLLMFIM(A/E)ML 158LDQLLLMFIMM(A/E)L 159 LDQLLL(A/E)FI(A/E)MML 160 LDQLLL(A/E)FIM(A/E)ML161 LDQLLL(A/E)FIMM(A/E)L 162 LDQLLLMFI(A/E)(A/E)ML 163LDQLLLMFI(A/E)M(A/E)L 164 LDQLLLMFIM(A/E)(A/E)L 165LDQLLLMFI(A/E)(A/E)(A/E)L 166 LDQLLL(A/E)FIM(A/E)(A/E)L 167LDQLLL(A/E)FI(A/E)M(A/E)L 168 LDQLLL(A/E)FI(A/E)(A/E)ML 169LDQLLL(A/E)FI(A/E)(A/E)(A/E)L 170 (E/V)DLLLMFIMMML 171(E/V)DLLL(A/E)FIMMML 172 (E/V)DLLLMFI(A/E)MML 173 (E/V)DLLLMFIM(A/E)ML174 (E/V)DLLLMFIMM(A/E)L 175 (E/V)DLLL(A/E)FI(A/E)MML 176(E/V)DLLL(A/E)FIM(A/E)ML 177 (E/V)DLLL(A/E)FIMM(A/E)L 178(E/V)DLLLMFI(A/E)(A/E)ML 179 (E/V)DLLLMFI(A/E)M(A/E)L 180(E/V)DLLLMFIM(A/E)(A/E)L 181 (E/V)DLLLMFI(A/E)(A/E)(A/E)L 182(E/V)DLLL(A/E)FIM(A/E)(A/E)L 183 (E/V)DLLL(A/E)FI(A/E)M(A/E)L 184(E/V)DLLL(A/E)FI(A/E)(A/E)ML 185 (E/V)DLLL(A/E)FI(A/E)(A/E)(A/E)L 186(E/V)DQLLLMFIMMML 187 (E/V)DQLLL(A/E)FIMMML 188 (E/V)DQLLLMFI(A/E)MML189 (E/V)DQLLLMFIM(A/E)ML 190 (E/V)DQLLLMFIMM(A/E)L 191(E/V)DQLLL(A/E)FI(A/E)MML 192 (E/V)DQLLL(A/E)FIM(A/E)ML 193(E/V)DQLLL(A/E)FIMM(A/E)L 194 (E/V)DQLLLMFI(A/E)(A/E)ML 195(E/V)DQLLLMFI(A/E)M(A/E)L 196 (E/V)DQLLLMFIM(A/E)(A/E)L 197(E/V)DQLLLMFI(A/E)(A/E)(A/E)L 198 (E/V)DQLLL(A/E)FIM(A/E)(A/E)L 199(E/V)DQLLL(A/E)FI(A/E)M(A/E)L 200 (E/V)DQLLL(A/E)FI(A/E)(A/E)ML 201(E/V)DQLLL(A/E)FI(A/E)(A/E)(A/E)L 202 LD(E/V)LLMFIMMML 203LD(E/V)LL(A/E)FIMMML 204 LD(E/V)LLMFI(A/E)MML 205 LD(E/V)LLMFIM(A/E)ML206 LD(E/V)LLMFIMM(A/E)L 207 LD(E/V)LL(A/E)FI(A/E)MML 208LD(E/V)LL(A/E)FIM(A/E)ML 209 LD(E/V)LL(A/E)FIMM(A/E)L 210LD(E/V)LLMFI(A/E)(A/E)ML 211 LD(E/V)LLMFI(A/E)M(A/E)L 212LD(E/V)LLMFIM(A/E)(A/E)L 213 LD(E/V)LLMFI(A/E)(A/E)(A/E)L 214LD(E/V)LL(A/E)FIM(A/E)(A/E)L 215 LD(E/V)LL(A/E)FI(A/E)M(A/E)L 216LD(E/V)LL(A/E)FI(A/E)(A/E)ML 217 LD(E/V)LL(A/E)FI(A/E)(A/E)(A/E)L 218LDQ(E/V)LLMFIMMML 219 LDQ(E/V)LL(A/E)FIMMML 220 LDQ(E/V)LLMFI(A/E)MML221 LDQ(E/V)LLMFIM(A/E)ML 222 LDQ(E/V)LLMFIMM(A/E)L 223LDQ(E/V)LL(A/E)FI(A/E)MML 224 LDQ(E/V)LL(A/E)FIM(A/E)ML 225LDQ(E/V)LL(A/E)FIMM(A/E)L 226 LDQ(E/V)LLMFI(A/E)(A/E)ML 227LDQ(E/V)LLMFI(A/E)M(A/E)L 228 LDQ(E/V)LLMFIM(A/E)(A/E)L 229LDQ(E/V)LLMFI(A/E)(A/E)(A/E)L 230 LDQ(E/V)LL(A/E)FIM(A/E)(A/E)L 231LDQ(E/V)LL(A/E)FI(A/E)M(A/E)L 232 LDQ(E/V)LL(A/E)FI(A/E)(A/E)ML 233LDQ(E/V)LL(A/E)FI(A/E)(A/E)(A/E)L 234 LDL(E/V)LMFIMMML 235LDL(E/V)L(A/E)FIMMML 236 LDL(E/V)LMFI(A/E)MML 237 LDL(E/V)LMFIM(A/E)ML238 LDL(E/V)LMFIMM(A/E)L 239 LDL(E/V)L(A/E)FI(A/E)MML 240LDL(E/V)L(A/E)FIM(A/E)ML 241 LDL(E/V)L(A/E)FIMM(A/E)L 242LDL(E/V)LMFI(A/E)(A/E)ML 243 LDL(E/V)LMFI(A/E)M(A/E)L 244LDL(E/V)LMFIM(A/E)(A/E)L 245 LDL(E/V)LMFI(A/E)(A/E)(A/E)L 246LDL(E/V)LL(A/E)FIM(A/E)(A/E)L 247 LDL(E/V)LL(A/E)FI(A/E)M(A/E)L 248LDL(E/V)LL(A/E)FI(A/E)(A/E)ML 249 LDL(E/V)L(A/E)FI(A/E)(A/E)(A/E)L 250LDQL(E/V)LMFIMMML 251 LDQL(E/V)L(A/E)FIMMML 252 LDQL(E/V)LMFI(A/E)MML253 LDQL(E/V)LMFIM(A/E)ML 254 LDQL(E/V)LMFIMM(A/E)L 255LDQL(E/V)L(A/E)FI(A/E)MML 256 LDQL(E/V)L(A/E)FIM(A/E)ML 257LDQL(E/V)L(A/E)FIMM(A/E)L 258 LDQL(E/V)LMFI(A/E)(A/E)ML 259LDQL(E/V)LMFI(A/E)M(A/E)L 260 LDQL(E/V)LMFIM(A/E)(A/E)L 261LDQL(E/V)LMFI(A/E)(A/E)(A/E)L 262 LDQL(E/V)LL(A/E)FIM(A/E)(A/E)L 263LDQL(E/V)LL(A/E)FI(A/E)M(A/E)L 264 LDQL(E/V)LL(A/E)FI(A/E)(A/E)ML 265LDQL(E/V)L(A/E)FI(A/E)(A/E)(A/E)L 266 LDLL(E/V)MFIMMML 267LDLL(E/V)(A/E)FIMMML 268 LDLL(E/V)MFI(A/E)MML 269 LDLL(E/V)MFIM(A/E)ML270 LDLL(E/V)MFIMM(A/E)L 271 LDLL(E/V)(A/E)FI(A/E)MML 272LDLL(E/V)(A/E)FIM(A/E)ML 273 LDLL(E/V)(A/E)FIMM(A/E)L 274LDLL(E/V)MFI(A/E)(A/E)ML 275 LDLL(E/V)MFI(A/E)M(A/E)L 276LDLL(E/V)MFIM(A/E)(A/E)L 277 LDLL(E/V)MFI(A/E)(A/E)(A/E)L 278LDLL(E/V)LL(A/E)FIM(A/E)(A/E)L 279 LDLL(E/V)LL(A/E)FI(A/E)M(A/E)L 280LDLL(E/V)LL(A/E)FI(A/E)(A/E)ML 281 LDLL(E/V)(A/E)FI(A/E)(A/E)(A/E)L 282LDQLL(E/V)MFIMMML 283 LDQLL(E/V)(A/E)FIMMML 284 LDQLL(E/V)MFI(A/E)MML285 LDQLL(E/V)MFIM(A/E)ML 286 LDQLL(E/V)MFIMM(A/E)L 287LDQLL(E/V)(A/E)FI(A/E)MML 288 LDQLL(E/V)(A/E)FIM(A/E)ML 289LDQLL(E/V)(A/E)FIMM(A/E)L 290 LDQLL(E/V)MFI(A/E)(A/E)ML 291LDQLL(E/V)MFI(A/E)M(A/E)L 292 LDQLL(E/V)MFIM(A/E)(A/E)L 293LDQLL(E/V)MFI(A/E)(A/E)(A/E)L 294 LDQLL(E/V)LL(A/E)FIM(A/E)(A/E)L 295LDQLL(E/V)LL(A/E)FI(A/E)M(A/E)L 296 LDQLL(E/V)LL(A/E)FI(A/E)(A/E)ML 297LDQLL(E/V)(A/E)FI(A/E)(A/E)(A/E)L 298 LDLLL(E/V)FIMMML 299LDLLL(E/V)FI(A/E)MML 300 LDLLL(E/V)FIM(A/E)ML 301 LDLLL(E/V)FIMM(A/E)L302 LDLLL(E/V)FI(A/E)(A/E)ML 303 LDLLL(E/V)FI(A/E)M(A/E)L 304LDLLL(E/V)FIM(A/E)(A/E)L 305 LDLLL(E/V)FI(A/E)(A/E)(A/E)L 306LDQLLL(E/V)FIMMML 307 LDQLLL(E/V)FI(A/E)MML 308 LDQLLL(E/V)FIM(A/E)ML309 LDQLLL(E/V)FIMM(A/E)L 310 LDQLLL(E/V)FI(A/E)(A/E)ML 311LDQLLL(E/V)FI(A/E)M(A/E)L 312 LDQLLL(E/V)FIM(A/E)(A/E)L 313LDQLLL(E/V)FI(A/E)(A/E)(A/E)L 314 LDLLLM(E/V)IMMML 315LDLLL(A/E)(E/V)IMMML 316 LDLLLM(E/V)I(A/E)MML 317 LDLLLM(E/V)IM(A/E)ML318 LDLLLM(E/V)IMM(A/E)L 319 LDLLL(A/E)(E/V)I(A/E)MML 320LDLLL(A/E)(E/V)IM(A/E)ML 321 LDLLL(A/E)(E/V)IMM(A/E)L 322LDLLLM(E/V)I(A/E)(A/E)ML 323 LDLLLM(E/V)I(A/E)M(A/E)L 324LDLLLM(E/V)IM(A/E)(A/E)L 325 LDLLL(A/E)(E/V)IM(A/E)(A/E)L 326LDLLL(A/E)(E/V)I(A/E)M(A/E)L 327 LDLLL(A/E)(E/V)I(A/E)(A/E)ML 328LDLLLM(E/V)I(A/E)(A/E)(A/E)L 329 LDQLLLM(E/V)IMMML 330LDQLLL(A/E)(E/V)IMMML 331 LDQLLLM(E/V)I(A/E)MML 332LDQLLLM(E/V)IM(A/E)ML 333 LDQLLLM(E/V)IMM(A/E)L 334LDQLLL(A/E)(E/V)I(A/E)MML 335 LDQLLL(A/E)(E/V)IM(A/E)ML 336LDQLLL(A/E)(E/V)IMM(A/E)L 337 LDQLLLM(E/V)I(A/E)(A/E)ML 338LDQLLLM(E/V)I(A/E)M(A/E)L 339 LDQLLLM(E/V)IM(A/E)(A/E)L 340LDQLLL(A/E)(E/V)IM(A/E)(A/E)L 341 LDQLLL(A/E)(E/V)I(A/E)M(A/E)L 342LDQLLL(A/E)(E/V)I(A/E)(A/E)ML 343 LDQLLLM(E/V)I(A/E)(A/E)(A/E)L 344LDLLLMF(E/V)MMML 345 LDLLL(A/E)F(E/V)MMML 346 LDLLLMF(E/V)(A/E)MML 347LDLLLMF(E/V)M(A/E)ML 348 LDLLLMF(E/V)MM(A/E)L 349LDLLL(A/E)F(E/V)(A/E)MML 350 LDLLL(A/E)F(E/V)M(A/E)ML 351LDLLL(A/E)F(E/V)MM(A/E)L 352 LDLLLMF(E/V)(A/E)(A/E)ML 353LDLLLMF(E/V)(A/E)M(A/E)L 354 LDLLLMF(E/V)M(A/E)(A/E)L 355LDLLLMF(E/V)(A/E)(A/E)(A/E)L 356 LDLLL(A/E)F(E/V)M(A/E)(A/E)L 357LDLLL(A/E)F(E/V)(A/E)M(A/E)L 358 LDLLL(A/E)F(E/V)(A/E)(A/E)ML 359LDLLL(A/E)F(E/V)(A/E)(A/E)(A/E)L 360 LDQLLLMF(E/V)MMML 361LDQLLL(A/E)F(E/V)MMML 362 LDQLLLMF(E/V)(A/E)MML 363LDQLLLMF(E/V)M(A/E)ML 364 LDQLLLMF(E/V)MM(A/E)L 365LDQLLL(A/E)F(E/V)(A/E)MML 366 LDQLLL(A/E)F(E/V)M(A/E)ML 367LDQLLL(A/E)F(E/V)MM(A/E)L 368 LDQLLLMF(E/V)(A/E)(A/E)ML 369LDQLLLMF(E/V)(A/E)M(A/E)L 370 LDQLLLMF(E/V)M(A/E)(A/E)L 371LDQLLLMF(E/V)(A/E)(A/E)(A/E)L 372 LDQLLL(A/E)F(E/V)M(A/E)(A/E)L 373LDQLLL(A/E)F(E/V)(A/E)M(A/E)L 374 LDQLLL(A/E)F(E/V)(A/E)(A/E)ML 375LDQLLL(A/E)F(E/V)(A/E)(A/E)(A/E)L 376 LDLLLMFI(E/V)MML 377LDLLL(A/E)FI(E/V)MML 378 LDLLLMFI(E/V)(A/E)ML 379 LDLLLMFI(E/V)M(A/E)L380 LDLLL(A/E)FI(E/V)(A/E)ML 381 LDLLL(A/E)FI(E/V)(A/E)ML 382LDLLL(A/E)FI(E/V)M(A/E)L 383 LDLLLMFI(E/V)(A/E)(A/E)L 384LDLLL(A/E)FI(E/V)(A/E)(A/E)L 385 LDQLLLMFI(E/V)MML 386LDQLLL(A/E)FI(E/V)MML 387 LDQLLLMFI(E/V)(A/E)ML 388LDQLLLMFI(E/V)M(A/E)L 389 LDQLLL(A/E)FI(E/V)(A/E)ML 390LDQLLL(A/E)FI(E/V)M(A/E)L 391 LDQLLLMFI(E/V)(A/E)(A/E)L 392LDQLLL(A/E)FI(E/V)(A/E)(A/E)L 393 LDLLLMFIM(E/V)ML 394LDLLL(A/E)FIM(E/V)ML 395 LDLLLMFIM(E/V)(A/E)L 396 LDLLLMFI(A/E)(E/V)ML397 LDLLL(A/E)FIM(E/V)(A/E)L 398 LDLLL(A/E)FI(A/E)(E/V)ML 399LDLLLMFI(A/E)(E/V)(A/E)L 400 LDLLL(A/E)FI(A/E)(E/V)(A/E)L 401LDQLLLMFIM(E/V)ML 402 LDQLLL(A/E)FIM(E/V)ML 403 LDQLLLMFIM(E/V)(A/E)L404 LDQLLLMFI(A/E)(E/V)ML 405 LDQLLL(A/E)FIM(E/V)(A/E)L 406LDQLLL(A/E)FI(A/E)(E/V)ML 407 LDQLLLMFI(A/E)(E/V)(A/E)L 408LDQLLL(A/E)FI(A/E)(E/V)(A/E)L 409 LDLLLMFIMM(E/V)L 410LDLLL(A/E)FIMM(E/V)L 411 LDLLLMFI(A/E)M(E/V)L 412 LDLLLMFIM(A/E)(E/V)L413 LDLLL(A/E)FI(A/E)M(E/V)L 414 LDLLL(A/E)FIM(A/E)(E/V)L 415LDLLLMFI(A/E)(A/E)(E/V)L 416 LDLLL(A/E)FI(A/E)(A/E)(E/V)L 417LDQLLLMFIMM(E/V)L 418 LDQLLL(A/E)FIMM(E/V)L 419 LDQLLLMFI(A/E)M(E/V)L420 LDQLLLMFIM(A/E)(E/V)L 421 LDQLLL(A/E)FI(A/E)M(E/V)L 422LDQLLL(A/E)FIM(A/E)(E/V)L 423 LDQLLLMFI(A/E)(A/E)(E/V)L 424LDQLLL(A/E)FI(A/E)(A/E)(E/V)L 425 LDLLLMFIMKM(E/V) 426LDLLL(A/E)FIMKM(E/V) 427 LDLLLMFI(A/E)MM(E/V) 428 LDLLLMFIM(A/E)M(E/V)429 LDLLLMFIMM(A/E)(E/V) 430 LDLLL(A/E)FI(A/E)MM(E/V) 431LDLLL(A/E)FIM(A/E)M(E/V) 432 LDLLL(A/E)FIMM(A/E)(E/V) 433LDLLLMFI(A/E)(A/E)M(E/V) 434 LDLLLMFI(A/E)M(A/E)(E/V) 435LDLLLMFIM(A/E)(A/E)(E/V) 436 LDLLLMFI(A/E)(A/E)(A/E)(E/V) 437LDLLL(A/E)FIM(A/E)(A/E)(E/V) 438 LDLLL(A/E)FI(A/E)M(A/E)(E/V) 439LDLLL(A/E)FI(A/E)(A/E)M(E/V) 440 LDLLL(A/E)FI(A/E)(A/E)(A/E)(E/V) 441LDQLLLMFIMMM(E/V) 442 LDQLLL(A/E)FIMMM(E/V) 443 LDQLLLMFI(A/E)MM(E/V)444 LDQLLLMFIM(A/E)M(E/V) 445 LDQLLLMFIMM(A/E)(E/V) 446LDQLLL(A/E)FI(A/E)MM(E/V) 447 LDQLLL(A/E)FIM(A/E)M(E/V) 448LDQLLL(A/E)FIMM(A/E)(E/V) 449 LDQLLLMFI(A/E)(A/E)M(E/V) 450LDQLLLMFI(A/E)M(A/E)(E/V) 451 LDQLLLMFIM(A/E)(A/E)(E/V) 452LDQLLLMFI(A/E)(A/E)(A/E)(E/V) 453 LDQLLL(A/E)FIM(A/E)(A/E)(E/V) 454LDQLLL(A/E)FI(A/E)M(A/E)(E/V) 455 LDQLLL(A/E)FI(A/E)(A/E)M(E/V) 456LDQLLL(A/E)FI(A/E)(A/E)(A/E)(E/V) 457 LKLLLMFIMMML 458 LKLLL(A/E)FIMMML459 LKLLLMFI(A/E)MML 460 LKLLLMFIM(A/E)ML 461 LKLLLMFIMM(A/E)L 462LKLLL(A/E)FI(A/E)MML 463 LKLLL(A/E)FIM(A/E)ML 464 LKLLL(A/E)FIMM(A/E)L465 LKLLLMFI(A/E)(A/E)ML 466 LKLLLMFI(A/E)M(A/E)L 467LKLLLMFIM(A/E)(A/E)L 468 LKLLLMFI(A/E)(A/E)(A/E)L 469LKLLL(A/E)FIM(A/E)(A/E)(E/V) 470 LKLLL(A/E)FI(A/E)M(A/E)(E/V) 471LKLLL(A/E)FI(A/E)(A/E)M(E/V) 472 LKLLL(A/E)FI(A/E)(A/E)(A/E)L 473LKQLLLMFIMMML 474 LKQLLL(A/E)FIMMML 475 LKQLLLMFI(A/E)MML 476LKQLLLMFIM(A/E)ML 477 LKQLLLMFIMM(A/E)L 478 LKQLLL(A/E)FI(A/E)MML 479LKQLLL(A/E)FIM(A/E)ML 480 LKQLLL(A/E)FIMM(A/E)L 481LKQLLLMFI(A/E)(A/E)ML 482 LKQLLLMFI(A/E)M(A/E)L 483LKQLLLMFIM(A/E)(A/E)L 484 LKQLLLMFI(A/E)(A/E)(A/E)L 485LKQLLL(A/E)FIM(A/E)(A/E)(E/V) 486 LKQLLL(A/E)FI(A/E)M(A/E)(E/V) 487LKQLLL(A/E)FI(A/E)(A/E)M(E/V) 488 LKQLLL(A/E)FI(A/E)(A/E)(A/E)L 489LD(E/S/A)LLLMFIMMML 490 LD(E/S/A)LLL(A/E)FIMMML 491LD(E/S/A)LLLMFI(A/E)MML 492 LD(E/S/A)LLLMFIM(A/E)ML 493LD(E/S/A)LLLMFIMM(A/E)L 494 LD(E/S/A)LLL(A/E)FI(A/E)MML 495LD(E/S/A)LLL(A/E)FIM(A/E)ML 496 LD(E/S/A)LLL(A/E)FIMM(A/E)L 497LD(E/S/A)LLLMFI(A/E)(A/E)ML 498 LD(E/S/A)LLLMFI(A/E)M(A/E)L 499LD(E/S/A)LLLMFIM(A/E)(A/E)L 500 LD(E/S/A)LLLMFI(A/E)(A/E)(A/E)L 501LD(E/S/A)LLL(A/E)FIM(A/E)(A/E)L 502 LD(E/S/A)LLL(A/E)FI(A/E)M(A/E)L 503LD(E/S/A)LLL(A/E)FI(A/E)(A/E)ML 504 LD(E/S/A)LLL(A/E)FI(A/E)(A/E)(A/E)L505

In certain embodiments, the peptide includes one or more mutationsrelative to the corresponding wildtype amino acid sequence ofSAGSEQQLDQLLAMFIMMMLQQ (SEQ ID NO: 7), which corresponds to amino acidresidues 33-54 of the HreX protein of Xanthomonas campestris pv.pelargonii (see U.S. Pat. No. 6,960,705, which is hereby incorporated byreference in its entirety). These one or more mutations include, inaddition to truncation of the full length, 114 aa HreX protein at bothits N-terminal and C-terminal ends, one or more deletions orsubstitutions relative to SEQ ID NO: 7. In certain embodiments, the oneor more mutations improve the solubility in aqueous solution, stability,and/or resistance to chemical degradation of the isolated peptiderelative to a polypeptide comprising or consisting of the correspondingwildtype amino acid sequence of SEQ ID NO:7. In this embodiment, theisolated peptide is stable when dissolved in water; resistant tochemical degradation in aqueous conditions in the presence of a pHbuffer and a biocide, as described above; and/or has a solubility in anaqueous solution of at least about 1.0 mg/ml. In alternativeembodiments, the one or more mutations disrupt the sequence of one ormore of the hydrophobic residues, and thereby alter the activity of thepeptide. For instance, in some embodiments, peptides will no longerinduce a hypersensitive response, but they will induce other activeplant responses including those described herein.

The isolated peptides of the invention can also be presented in the formof a fusion peptide that includes, in addition, a second amino acidsequence coupled to the inventive peptides via peptide bond. The secondamino acid sequence can be a purification tag, such as poly-histidine(His₆-), a glutathione-S-transferase (GST-), or maltose-binding protein(MBP-), which assists in the purification but can later be removed,i.e., cleaved from the peptide following recovery. Protease-specificcleavage sites or chemical-specific cleavage sites (i.e., in a cleavablelinker sequence) can be introduced between the purification tag and thedesired peptide. Protease-specific cleavage sites are well known in theliterature and include, without limitation, the enterokinase specificcleavage site (Asp)₄-Lys (SEQ ID NO: 547), which is cleaved afterlysine; the factor Xa specific cleavage site Ile-(Glu or Asp)-Gly-Arg(SEQ ID NO: 548), which is cleaved after arginine; the trypsin specificcleavage site, which cleaves after Lys and Arg; and the Genenase™ Ispecific cleavage site Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO: 549).Chemicals and their specific cleavage sites include, without limitation,cyanogen bromide (CNBr), which cleaves at methionine (Met) residues;BNPS-skatole, which cleaves at tryptophan (Trp) residues; formic acid,which cleaves at aspartic acid-proline (Asp-Pro) peptide bonds;hydroxylamine, which cleaves at asparagine-glycine (Asn-Gly) peptidebonds; and 2-nitro-5-thiocyanobenzoic acid (NTCB), which cleaves atcysteine (Cys) residues (see Crimmins et al., “Chemical Cleavage ofProteins in Solution,” Curr. Protocol. Protein Sci., Chapter 11:Unit11.4 (2005), which is hereby incorporated by reference in its entirety).In order to use one of these cleavage methods, it may be necessary toremove unwanted cleavage sites from within the desired peptide sequencesby mutation. For example, P5-3 is a mutant sequence derived from P5 withthe methionine residues mutated to alanine. Peptides comprising thissequence can be produced by cyanogen bromide-mediated cleavage of atandem repeated sequence of P5-3 separated by methionine residues.

The isolated peptides of the invention can also be presented in the formof a fusion peptide that includes multiple peptide sequences of thepresent invention linked together by a linker sequence, which may or maynot take the form of a cleavable amino acid sequence of the typedescribed above. Such multimeric fusion proteins may or may not includepurification tags. In one embodiment, each monomeric sequence caninclude a purification tag linked to a peptide of the invention by afirst cleavable peptide sequence; and the several monomeric sequencescan be linked to adjacent monomeric sequences by a second cleavablepeptide sequence. Consequently, upon expression of the multimeric fusionprotein, i.e., in a host cell, the recovered fusion protein can betreated with a protease or chemical that is effective to cleave thesecond cleavable peptide sequence, thereby releasing individualmonomeric peptide sequences containing purification tags. Upon affinitypurification, the recovered monomeric peptide sequences can be treatedwith a protease or chemical that is effective to cleave the firstcleavable peptide sequence and thereby release the purification tag fromthe peptide of interest. The latter can be further purified using gelfiltration and/or HPLC as described infra.

According to one approach, the peptides of the present invention can besynthesized by standard peptide synthesis operations. These include bothFMOC (9-fluorenylmethyloxy-carbonyl) and tBoc (tert-butyloxy-carbonyl)synthesis protocols that can be carried out on automated solid phasepeptide synthesis instruments including, without limitation, the AppliedBiosystems 431A, 433A synthesizers and Peptide Technologies Symphony orlarge scale Sonata or CEM Liberty automated solid phase peptidesynthesizers. The use of alternative peptide synthesis instruments isalso contemplated. Peptides prepared using solid phase synthesis arerecovered in a substantially pure form.

The peptides of the present invention may be also prepared by usingrecombinant expression systems followed by separation and purificationof the recombinantly prepared peptides. Generally, this involvesinserting an encoding nucleic acid molecule into an expression system towhich the molecule is heterologous (i.e., not normally present). One ormore desired nucleic acid molecules encoding a peptide of the inventionmay be inserted into the vector. The heterologous nucleic acid moleculeis inserted into the expression system or vector in proper sense (5′43′)orientation and correct reading frame relative to the promoter and anyother 5′ and 3′ regulatory molecules.

Representative nucleotide sequences for expression in representativebacteria and plant hosts are included in Table 3 below:

TABLE 3 Peptide & Optimized SEQ Host Nucleotide Sequence ID NO: P5 inAGCGCAGGTAGCGAACAGCAGCTGGATCTGCT 506 E. coliGCTGATGTTTATTATGATGATGCTGCAGCAG P5-21 inAGCGAAGAACAGCTGGAACTGCTGCTGGCATT 507 E. coliTATTGCAGCAGCACTGCAGCAGGAAGAA P5 in TCCGCCGGCTCCGAGCAGCAGCTGGACCTGCT 508Zea mays GCTGATGTTCATCATGATGATGCTGCAGCAG P5-21 inTCCGAGGAGCAGCTGGAGCTGCTGCTGGCCTT 509 Zea maysCATCGCCGCCGCCCTGCAGCAGGAGGAGWith knowledge of the encoded amino acid sequence listed herein and thedesired transgenic organism, additional codon-optimized DNA sequencesand RNA sequences can be generated with nothing more than routine skill.

Expression (including transcription and translation) of a peptide orfusion polypeptide of the invention by the DNA construct may beregulated with respect to the level of expression, the tissue type(s)where expression takes place and/or developmental stage of expression. Anumber of heterologous regulatory sequences (e.g., promoters andenhancers) are available for controlling the expression of the DNAconstruct in plants. These include constitutive, inducible andregulatable promoters, as well as promoters and enhancers that controlexpression in a tissue- or temporal-specific manner. Exemplaryconstitutive promoters include the raspberry E4 promoter (U.S. Pat. Nos.5,783,393 and 5,783,394, each of which is hereby incorporated byreference in its entirety), the nopaline synthase (NOS) promoter (Ebertet al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5745-5749 (1987), which ishereby incorporated by reference in its entirety), the octopine synthase(OCS) promoter (which is carried on tumor-inducing plasmids ofAgrobacterium tumefaciens), the caulimovirus promoters such as thecauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Mol.Biol. 9:315-324 (1987), which is hereby incorporated by reference in itsentirety) and the CaMV 35S promoter (Odell et al., Nature 313:810-812(1985), which is hereby incorporated by reference in its entirety), thefigwort mosaic virus 35S-promoter (U.S. Pat. No. 5,378,619, which ishereby incorporated by reference in its entirety), the light-induciblepromoter from the small subunit of ribulose-1,5-bis-phosphatecarboxylase (ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl.Acad. Sci. (U.S.A.) 84:6624-6628 (1987), which is hereby incorporated byreference in its entirety), the sucrose synthase promoter (Yang et al.,Proc. Natl. Acad. Sci. (U.S.A.) 87:4144-4148 (1990), which is herebyincorporated by reference in its entirety), the R gene complex promoter(Chandler et al., Plant Cell 1:1175-1183 (1989), which is herebyincorporated by reference in its entirety), the chlorophyll a/b bindingprotein gene promoter, the CsVMV promoter (Verdaguer et al., Plant MolBiol., 37:1055-1067 (1998), which is hereby incorporated by reference inits entirety), and the melon actin promoter (PCT Publ. No. WO00/56863,which is hereby incorporated by reference in its entirety). Exemplarytissue-specific promoters include the tomato E4 and E8 promoters (U.S.Pat. No. 5,859,330, which is hereby incorporated by reference in itsentirety) and the tomato 2AII gene promoter (Van Haaren et al., PlantMol Bio., 21:625-640 (1993), which is hereby incorporated by referencein its entirety).

In one preferred embodiment, expression of the DNA construct is undercontrol of regulatory sequences from genes whose expression isassociated with early seed and/or embryo development. Indeed, in apreferred embodiment, the promoter used is a seed-enhanced promoter.Examples of such promoters include the 5′ regulatory regions from suchgenes as napin (Kridl et al., Seed Sci. Res. 1:209:219 (1991), which ishereby incorporated by reference in its entirety), globulin (Belangerand Kriz, Genet. 129: 863-872 (1991), GenBank Accession No. L22295, eachof which is hereby incorporated by reference in its entirety), gammazein Z 27 (Lopes et al., Mol Gen Genet. 247:603-613 (1995), which ishereby incorporated by reference in its entirety), L3 oleosin promoter(U.S. Pat. No. 6,433,252, which is hereby incorporated by reference inits entirety), phaseolin (Bustos et al., Plant Cell 1(9):839-853 (1989),which is hereby incorporated by reference in its entirety), arcelin5(U.S. Application Publ. No. 2003/0046727, which is hereby incorporatedby reference in its entirety), a soybean 7S promoter, a 7Sa promoter(U.S. Application Publ. No. 2003/0093828, which is hereby incorporatedby reference in its entirety), the soybean 754 conglycinin promoter, a75α promoter (Beachy et al., EMBO J. 4:3047 (1985); Schuler et al.,Nucleic Acid Res. 10(24):8225-8244 (1982), each of which is herebyincorporated by reference in its entirety), soybean trypsin inhibitor(Riggs et al., Plant Cell 1(6):609-621 (1989), which is herebyincorporated by reference in its entirety), ACP (Baerson et al., PlantMol. Biol., 22(2):255-267 (1993), which is hereby incorporated byreference in its entirety), stearoyl-ACP desaturase (Slocombe et al.,Plant Physiol. 104(4):167-176 (1994), which is hereby incorporated byreference in its entirety), soybean a′ subunit of β-conglycinin (Chen etal., Proc. Natl. Acad. Sci. 83:8560-8564 (1986), which is herebyincorporated by reference in its entirety), Vicia faba USP (U.S.Application Publ. No. 2003/229918, which is hereby incorporated byreference in its entirety) and Zea mays L3 oleosin promoter (Hong etal., Plant Mol. Biol., 34(3):549-555 (1997), which is herebyincorporated by reference in its entirety).

Nucleic acid molecules encoding the peptides of the present inventioncan be prepared via solid-phase synthesis using, e.g., thephosphoramidite method and phosphoramidite building blocks derived fromprotected 2′-deoxynucleosides. To obtain the desired oligonucleotide,the building blocks are sequentially coupled to the growingoligonucleotide chain in the order required by the sequence of theproduct. Upon the completion of the chain assembly, the product isreleased from the solid phase to solution, deprotected, collected, andtypically purified using HPLC. The limits of solid phase synthesis aresuitable for preparing oligonucleotides up to about 200 nt in length,which encodes peptides on the order of about 65 amino acids or less. Theends of the synthetized oligonucleotide can be designed to includespecific restriction enzyme cleavage site to facilitate ligation of thesynthesized oligonucleotide into an expression vector.

For longer peptides, oligonucleotides can be prepared via solid phasesynthesis and then the synthetic oligonucleotide sequences ligatedtogether using various techniques. Recombinant techniques for thefabrication of whole synthetic genes are reviewed, for example, inHughes et al., “Chapter Twelve—Gene Synthesis: Methods andApplications,” Methods in Enzymology 498:277-309 (2011), which is herebyincorporated by reference in its entirety.

Synthetic oligonucleotides of the present invention include both DNA andRNA, in both D and L enantiomeric forms, as well as derivatives thereof(including, but not limited to, 2′-fluoro-, 2′-amino, 2′O-methyl,5′iodo-, and 5′-bromo-modified polynucleotides). Nucleic acidscontaining modified nucleotides (Kubik et al., “Isolation andCharacterization of 2′fluoro-, 2′ amino-, and 2′fluoro-amino-modifiedRNA Ligands or Human IFN-gamma that Inhibit Receptor Binding,” J.Immunol. 159:259-267 (1997); Pagratis et al., “Potent 2′-amino, and2′-fluoro-2′-deoxy-ribonucleotide RNA Inhibitors of Keratinocyte GrowthFactor,” Nat. Biotechnol. 15:68-73 (1997), each which is herebyincorporated by reference in its entirety) and the L-nucleic acids(sometimes termed Spiegelmers®), enantiomeric to natural D-nucleic acids(Klussmann et al., “Mirror-image RNA that Binds D-adenosine,” Nat.Biotechnol. 14:1112-1115 (1996) and Williams et al., “Bioactive andnuclease-resistant L-DNA Ligand of Vasopressin,” Proc. Natl. Acad. Sci.USA 94:11285-11290 (1997), each which is hereby incorporated byreference in its entirety), and non-natural bases are used to enhancebiostability. In addition, the sugar-phosphate backbone can be replacedwith a peptide backbone, forming a peptide nucleic acid (PNA), othernatural or non-natural sugars can be used (e.g., 2′-deoxyribose sugars),or phosphothioate or phosphodithioate can be used instead ofphosphodiester bonds. The use of locked nucleic acids (LNA) is alsocontemplated. These nucleic acid molecules can be used for multiplepurposes, including application to plants or plants seeds as nakedoligonucleotides or for in vitro translation of encodingoligonucleotides for production of the peptides of the presentinvention.

Once a suitable expression vector is selected, the desired nucleic acidsequences are cloned into the vector using standard cloning proceduresin the art, as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y.(1989), or U.S. Pat. No. 4,237,224 to Cohen and Boyer, which are herebyincorporated by reference in their entirety. The vector is thenintroduced to a suitable host.

A variety of host-vector systems may be utilized to recombinantlyexpress the peptides of the present invention. Primarily, the vectorsystem must be compatible with the host used. Host-vector systemsinclude, without limitation, the following: bacteria transformed withbacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such asyeast containing yeast vectors; mammalian cell systems infected withvirus (e.g., vaccinia virus, adenovirus, etc.); insect cell systemsinfected with virus (e.g., baculovirus); and plant cells infected byAgrobacterium. The expression elements of these vectors vary in theirstrength and specificities. Depending upon the host-vector systemutilized, any one of a number of suitable transcription and translationelements can be used to carry out this and other aspects of the presentinvention.

Purified peptides may be obtained by several methods. The peptide ispreferably produced in purified form (preferably at least about 80% or85% pure, more preferably at least about 90% or 95% pure) byconventional techniques. Depending on whether the recombinant host cellis made to secrete the peptide into growth medium (see U.S. Pat. No.6,596,509 to Bauer et al., which is hereby incorporated by reference inits entirety), the peptide can be isolated and purified bycentrifugation (to separate cellular components from supernatantcontaining the secreted peptide) followed by sequential ammonium sulfateprecipitation of the supernatant. The fraction containing the peptide issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the peptides from other proteins. Ifnecessary, the peptide fraction may be further purified by HPLC.

Alternatively, if the peptide of interest of interest is not secreted,it can be isolated from the recombinant cells using standard isolationand purification schemes. This includes disrupting the cells (e.g., bysonication, freezing, French press, etc.) and then recovering thepeptide from the cellular debris. Purification can be achieved using thecentrifugation, precipitation, and purification procedures describedabove. The use of purification tags, described above, can simplify thisprocess.

In certain embodiments, purification is not required. Where purificationis not performed, cell-free lysates can be recovered followingcentrifugation for removal of cellular debris. The resulting cell-freelysate can be treated with heat for a sufficient amount of time todeactivate any native proteases in the recovered fraction, e.g., 10 minat 100° C. If desired, one or more of biocidal agents, proteaseinhibitors, and non-ionic surfactants can be introduced to such acell-free preparation (see U.S. Application Publ. No. 20100043095 toWei, which is hereby incorporated by reference in its entirety).

Once the peptides of the present invention are recovered, they can beused to prepare a composition that includes a carrier, and one or moreadditives selected from the group consisting of a bacteriocidal orbiocidal agent, a protease inhibitor, a non-ionic surfactant, afertilizer, an herbicide, an insecticide, a fungicide, a nematicide,biological inoculants, plant regulators, and mixtures thereof.

In certain embodiments, the compositions include greater than about 1 nMof the peptide, greater than about 10 nM of the peptide, greater thanabout 20 nM of the peptide, greater than about 30 nM of the peptide,greater than about 40 nM of the peptide, greater than about 50 nM of thepeptide, greater than about 60 nM of the peptide, greater than about 70nM of the peptide, greater than 80 about nM of the peptide, greater thanabout 90 nM of the peptide, greater than about 100 nM of the peptide,greater than about 150 nM of the peptide, greater than about 200 nM ofthe peptide, or greater than about 250 nM of the peptide. In certainembodiments, the compositions include less than about 1 nM of thepeptide. For example, certain peptides can be present at a concentrationof less than about 2 ng/ml, less than about 1.75 ng/ml, less than about1.5 ng/ml, less than about 1.25 ng/ml, less than about 1.0 ng/ml, lessthan about 0.75 ng/ml, less than about 0.5 ng/ml, less than about 0.25ng/ml, or even less than about 0.1 ng/ml.

Suitable carriers include water, aqueous solutions optionally containingone or more co-solvents, slurries, and solid carrier particles.Exemplary solid carriers include mineral earths such as silicates,silica gels, talc, kaolins, limestone, lime, chalk, bole, loess, clays,dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate,magnesium oxide, ground synthetic materials, and products of vegetableorigin, such as cereal meal, tree bark meal, wood meal and nutshellmeal, cellulose powders, starches and starch derivatives, as well asother mono-, di-, and poly-saccharides.

Suitable fertilizers include, without limitation, ammonium sulfate,ammonium phosphate, ammonium nitrate, ureas, and combinations thereof.

Suitable insecticides include, without limitation, members of theneonicotinoid class such as imidicloprid, clothianidin, andthiamethoxam; members of the organophosphate class such as chlorpyrifosand malathion; members of the pyrethroid class such as permethrin; othernatural insecticides such as nicotine, nornicotine, and pyrethrins;members of the carbamate class such as aldicarb, carbofuran, andcarbaryl; members of the macrocyclic lactone class such as variousabamectin, avermectin, and ivermectin products; members of the diamideclass such as chlorantraniliprole, cyantraniliprole, and flubendiamide;chitin synthesis inhibitors, particularly those of the benzoylurea classsuch as lufenuron and diflubenzuron; and any combination thereof,including combinations of two or more, three or more, or four or moreinsecticides. Additional insecticides are listed in the Compendium ofPesticide Common Names, which is database operated by Alan Wood andavailable in electronic form at the alanwood.net internet site.

Suitable fungicides include, without limitation, members of thestrobilurin class such as azoxystrobin, pyraclostrobin, trifloxystrobin,picoxystrobin, and fluoxastrobin; members of the triazole class such asipconazole, metconazole, tebuconazole, triticonazole, tetraconazole,difenoconazole, flutriafol, propiconazole and prothioconazole; membersof the succinate dehydrogenase class such as carboxin, fluxapyroxad,boscalid and sedaxane: members of the phenylamide class such asmetalaxyl, mefenoxam, benalaxyl, and oxadiyxl; members of thephenylpyrrole class such as fludioxonil; members of the phthalimideclass such as captan; members of the dithiocarbamate class such asmancozeb and thiram; members of the benzimidazole class such asthiabendazole; and any combination thereof, including combinations oftwo or more, three or more, or four or more fungicides. Additionalfungicides are listed in the Compendium of Pesticide Common Names, whichis a database operated by Alan Wood and available in electronic form atthe alanwood.net internet site.

Suitable nematicides include, without limitation, chemicals of thecarbamate class such as aldicarb, aldoxycarb, oxamyl, carbofuran, andcleothocarb; and chemicals of the organophosphate class such asthionazin, ethoprophos, fenamiphos, fensulfothion, terbufos, isazofos,and ebufos. Additional nematicides are listed in the Compendium ofPesticide Common Names, which is a database operated by Alan Wood andavailable in electronic form at the alanwood.net internet site.

Suitable bactericides include, without limitation, those based ondichlorophene and benzylalcohol hemi formal (Proxel® from ICI orActicide® RS from Thor Chemie and Kathon® MK from Rohm & Haas) andisothiazolinone derivatives such as alkylisothiazolinones andbenzisothiazolinones (Acticide® MBS from Thor Chemie; Proxel® GXL fromICI). Additional bactericides are listed in the Compendium of PesticideCommon Names, which is a database operated by Alan Wood and available inelectronic form at the alanwood.net internet site.

Suitable inoculants include, without limitation, Bradyrhizobium spp.,particularly Bradyrhizobium japonicum (BASF Vault® products), Bacillussubtilis, Bacillus firmus, Bacillus pumilis, Streptomyces lydicus,Trichoderma spp., Pasteuria spp., other cultures of rhizobial cells(BASF Nodulator® and Rhizo-Flo®), and any combination thereof, includingcombinations of two or more, three or more, or four or more inoculants.The inoculants can be recombinant in nature, as described hereinafter,to facilitate expression and optionally secretion of a polypeptide ofthe invention. Alternatively, these inoculants can be otherwisecommercially available forms that are unable to express/secrete apolypeptide of the invention.

Plant regulators are chemical substances, whether natural or synthetic,that either stimulate or inhibit plant biochemical signaling. These areusually, but not exclusively, recognized by receptors on the surface ofthe cell, causing a cascade of reactions in the cell. Suitable plantregulators include, without limitation, ethephon; ethylene; salicylicacid; acetylsalicylic acid; jasmonic acid; methyl jasmonate; methyldihydrojasmonate; chitin; chitosan; abscisic acid; any auxin compound orinhibitor, including but not limited to (4-chlorophenoxy)acetic acid,(2,4-dichlorophenoxy)acetic acid, and 2,3,5-triiodobenzoic acid; anycytokinin, including but not limited to kinetin and zeatin;gibberellins; brassinolide; and any combination thereof, includingcombinations of two or more, three or more, or four or more regulators.

Other suitable additives include buffering agents, wetting agents,coating agents, and abrading agents. These materials can be used tofacilitate application of the compositions in accordance with thepresent invention. In addition, the compositions can be applied to plantseeds with other conventional seed formulation and treatment materials,including clays and polysaccharides.

Compositions or systems use for plant seed treatment include: one ormore of the peptides of the present invention, preferably though notexclusively one of P5, P5-8, P5-21, P5-25, P5-27, P5-35, or P5-46 (SEQID NOS: 8, 14, 33, 52, 53, 71, and 58 respectively) in combination withone or more insecticides, nematicides, fungicides, other inoculants, orother plant regulators, including combinations of multiple insecticides,or multiple nematicides, multiple fungicides, multiple other inoculants,or multiple plant regulators. Suitable insecticides, nematicides,fungicides, inoculants, and plant regulators for these combinationtreatments include those identified above. These compositions arepresented in the form of a single composition at the time of seedtreatment. In contrast, a system used for seed treatment may involvemultiple treatments, e.g., a composition containing the peptides is usedin one treatment and a composition containing the one or moreinsecticides, nematicides, fungicides, plant regulators and/orbactericides, is used in a separate treatment. In the latter embodiment,both of these treatments are carried out at about the same time, i.e.,before planting or at about the time of planting.

One such example includes one or more of peptides of the presentinvention, including (without limitation) one of P5, P5-8, P5-21, P5-25,P5-27, P5-35, or P5-46 (SEQ ID NOS: 8, 14, 33, 52, 53, 71, and 58respectively), in combination with Poncho™ (clothianidin) available fromBayer Crop Science, Poncho™ VOTiVO (clothianidin and Bacillus firmusbiological nematicide) available from Bayer Crop Science, and Gaucho™(imidicloprid) available from Bayer Crop Science.

Another example includes one or more of peptides of the presentinvention, including (without limitation) one of P5, P5-8, P5-21, P5-25,P5-27, P5-35, or P5-46 (SEQ ID NOS: 8, 14, 33, 52, 53, 71, and 58respectively), in combination with Cruiser™ (thiamethoxam) availablefrom Syngenta, CruiserMaxx™ (thiamethoxam, mefenoxam, and fludioxynil)available from Syngenta, Cruiser Extreme™ (thiamethoxam, mefenoxam,fludioxynil, and azoxystrobin) available from Syngenta, Avicta™(thiamethoxam and abamectin) available from Syngenta, and Avicta™Complete (thiamethoxam, abamectin, and Clariva Complete™ which containsthe Pasteuria nishizawae—Pn1 biological inoculant) available fromSyngenta, and Avicta Complete™ Corn (thiamethoxam, mefenoxam,fludioxynil, azoxystrobin, thiabendazole and abamectin) available fromSyngenta.

Another example includes one or more of peptides of the presentinvention, including (without limitation) one of P5, P5-8, P5-21, P5-25,P5-27, P5-35, or P5-46 (SEQ ID NOS: 8, 14, 33, 52, 53, 71, and 58respectively), in combination with Vault Liquid plus Integral(Bradyrhizobium species and Bacillus subtilis strain MBI 600 inoculants)available from BASF, Vault NP (Bradyrhizobium japonicum inoculant)available from BASF, and Subtilex NG (Bacillus subtilis biologicalinoculant) available from BASF.

As an alternative to using peptides or compositions to apply thepeptides of the present invention to plants, the use of beneficialmicrobes to deliver the peptide to the plant or plant seed, or the locuswhere the plant seed is planted in soil (and where the mature plant isgrown), is also contemplated. Thus, a further aspect of the inventioninvolves the engineering and application of beneficial microbes toproduce peptides of the present invention. Beneficial microbes execute anumber of useful activities, reviewed in Glick, “Plant Growth-PromotingBacteria: Mechanisms and Applications,” Scientifica, Article ID 963401(2012), which is hereby incorporated by reference in its entirety.Beneficial microbes can provide nutrition to a plant. This may come inthe form of amino acids and other nitrogen-containing compounds throughthe process of nitrogen fixation. Beneficial microbes may also liberatephosphate from inaccessible mineral deposits in the soil and make theseavailable. For example, bacteria can synthesize siderophores which bindand solubilize inaccessible iron deposits. These iron-siderophorecomplexes can be absorbed by plants. Microbes can produce analogs ofplant signaling hormones which stimulate growth and reduce stresssignaling. Finally, beneficial microbes can compete with pathogenicorganisms by removing resources including iron as well as synthesis ofantibiotic compounds. Beneficial microbes may exhibit other behaviorsand are not limited to the behaviors listed above. Beneficial organismsare classified as epiphytic (living on or near the surface of planttissues) or endophytic (living within plant tissues).

Suitable beneficial bacterium include, without limitation, Pseudomonas(e.g., P. fluorescens, P. aureofaciens, P. chlororaphis, P.solanacearum, and P. syringae), Sphingomonas (e.g., S. phyllosphaerae,S. roseiflava, S. melonis, S. azotifigens, and S. mali) (see alsoInnerebner et al., “Protection of Arabidopsis thaliana AgainstLeaf-Pathogenic Pseudomonas syringae by Sphingomonas Strains in aControlled Model System,” Appl. Environ. Microbiol. 77:3202-3210 (2011),which is hereby incorporated by reference in its entirety), Bacillus (B.firmus, B. licheniformis, B. megaterium, B. mucilaginous, B. pumilus, B.subtilis, and B. subtilis var. amyloliquefaciens), Streptomyces (e.g.,S. griseoviridis and S. lydicus), Rhizobium (e.g., R. meliloti, R.trifolii, R. leguminosarum, R. phaseolin, R. lupine, and R. japonicum),Frankia (e.g., F. alni), and Azospirillum (e.g., A. brasilense and A.lipoferum).

Additional beneficial bacterium, include, without limitation,Agrobacterium radiobacter, Azotobacter chroococcum, Burkholderiacepacia, Delfitia acidovorans, Paenobacillus macerans, Pantoeaagglomerans, and Serratia entomophilia.

In certain embodiments, the beneficial microbe may be a filamentousfungal host cell. In some embodiments, the host cell may be a cell of astrain that has a history of use for production of proteins that hasGRAS status, i.e., a Generally Recognized as Safe, by the FDA.

In some embodiments, beneficial fungal microbes may be of a strain ofAspergillus niger which include ATCC 22342, ATCC 44733, ATCC 14331, ATCC11490, NRRL 3112, and strains derived therefrom. In some embodiments,beneficial fungal microbes may be strains of Trichoderma (e.g. T.harzianum, T. viride, T. koningi, T. reesei and T. hamatum) whichinclude functional equivalents of RL-P37 (Sheir-Neiss et al. (1984)Appl. Microbiol. Biotechnology 20:46-53, which is hereby incorporated byreference in its entirety). Other useful beneficial fungal microbesinclude, without limitation, NRRL 15709, ATCC 13631, ATCC 26921 (QM9414) ATCC 32098, ATCC 32086, and ATCC 56765 (RUT-30). In someembodiments, beneficial fungal microbes may be strains ofnon-filamentous fungal yeasts, including, without limitation, strains ofRhodotorula (e.g., R. graminis WP1 and R. mucilaginosa) (see U.S. Pat.No. 8,728,781 and Xin et al., “Characterization of Three Endophytic,Indole-3-Acetic Acid-Producing Yeasts Occurring in Populus Trees,”Mycol. Res. 113:973-980 (2009), which are hereby incorporated byreference in their entirety).

Peptide expression systems can be created using existing plasmid systemsby one skilled in the art. One notable guideline is that regulation ofpeptide expression should be well controlled. High peptideconcentrations detected by the plant will likely trigger an intenseimmune response with widespread cell death characteristic of thehypersensitive response. In contrast, lower peptide expression levelsshould stimulate the immunity while minimizing cell death. This effectmay be further balanced by careful choice of secretion sequences.Expression of peptides in Pseudomonas fluorescens may be accomplishedusing the expression strains and tools described by Retallack et al.,“Reliable protein production in a Pseudomonas fluorescens expressionsystem,” Protein Expression and Purification 81:157-65 (2012), which ishereby incorporated by reference in its entirety. Expression of peptidesin Bacillus subtilis can be accomplished through vectors utilizing asubtilisin (aprE) promoter system. This can optionally be augmentedusing signal peptides to direct secretion of the peptide outside of themicrobe. These functions are implemented in the “Bacillus SubtilisSecretory Protein Expression System” manual available from Clontech.Expression of proteins in Streptomyces has been demonstrated usingplasmids as described by Fernandez-Abalos et al., “Posttranslationalprocessing of the xylanase Xys1L from Streptomyces halstedii JM8 iscarried out by secreted serine proteases,” Microbiology 149:1623-32(2003), which is hereby incorporated by reference in its entirety.Additional peptide expression systems can be produced by one skilled inthe art.

Once engineered microbes are raised, e.g., in a fermentation apparatus,the engineered microbes can be recovered and then provided in either adry composition or a liquid composition or suspension. For liquidcompositions or suspensions, the microbes can be mixed in water, or abuffer solution, and applied as a spray treatment to the plants or thelocus where plants are grown. Alternatively, the solution can be used asa seed treatment prior to planting the seeds. For dry compositions, themicrobes can be dried with or without inert carrier particles, and thedry composition can be applied to seeds, the locus where seeds will beplanted or plants are being grown, or directly to plants.

Colony forming units (c.f.u.) are used to quantify microbes. 1 c.f.u. ofa microbe generates a single colony when spread onto a solid nutrientagar compatible with the organism and corresponds to one healthy,replication competent cell. In a dry powder formulation, theconcentration of microbes can exceed 5×10¹⁰ cfu/gram of material.Suitable concentrations for a dry formulation include >10¹¹, >5×10¹⁰,>10¹⁰, >10⁹, >10⁸, 10⁷, or >10⁶ cfu/gram. Likewise, microbes can beprovided as a liquid suspension. Suitable concentrations for a liquidformulation include >10¹⁰, >10⁹, >10⁸, >10⁷, >10⁶, >10⁵ cfu/ml.

The present invention further relates to methods of imparting diseaseresistance to plants, enhancing plant growth, effecting pest control,imparting biotic or abiotic stress tolerance to plants, and/ormodulating plant biochemical signaling. According to one embodiment,these methods involve applying an effective amount of an isolatedpeptide of the invention, or a composition of the invention to a plantor plant seed or the locus where the plant is growing or is expected togrow. As a consequence of such application, the peptide contacts cellsof the plant or plant seed, and induces in the plant or a plant grownfrom the plant seed disease resistance, growth enhancement, tolerance tobiotic stress, tolerance to abiotic stress, or altered biochemicalsignaling. According to an alternative embodiment, the peptide orcomposition of the invention can be applied to plants such that seedsrecovered from such plants themselves are able to impart diseaseresistance in plants, to enhance plant growth, to affect insect control,to impart tolerance to biotic or abiotic stress, and/or to modulatebiochemical signaling, to modulate maturation. According to yet anotherembodiment, these methods involve applying a recombinant inoculant tothe plant seeds or plants, or the locus where the plant is growing or isexpected to grow. As a consequence of such application, the recombinantinoculant expresses or secretes a peptide of the invention and thepeptide contacts cells of the plant or plant seeds and induces in theplant or a plant grown from the plant seed disease resistance, growthenhancement, tolerance to biotic stress, tolerance to abiotic stress, oraltered biochemical signaling.

In these embodiments, it is also possible to select plants or plantseeds or the locus to which the isolated peptide or composition of theinvention is applied. For example, for fields known to contain a highnematode content, the plants or plant seeds to be grown in such fields,or the fields (locus), can be selectively treated by applying theisolated peptide or composition or recombinant inoculant of theinvention as described herein; whereas no such treatment may benecessary for plants or plant seeds grown in fields containing lownematode content. Similarly, for fields having reduced irrigation, theplants or plant seeds to be grown in such fields, or the fields (locus),can be selectively treated by applying the isolated peptide orcomposition or recombinant inoculant of the invention as describedherein; whereas no such treatment may be necessary for plants or plantseeds grown in fields having adequate irrigation. Likewise, for fieldsprone to flooding, the plants or plant seeds to be grown in such fields,or the fields (locus), can be selectively treated by applying theisolated peptide or composition or recombinant inoculant of theinvention as described herein; whereas no such treatment may benecessary for plants or plant seeds grown in fields that are not proneto flooding. As yet another example of such selection, for fields proneto insect attack at certain times of the growing season, the plants orplant seeds to be grown in such fields, or the fields (locus), can beselectively treated by applying the isolated peptide or composition ofthe invention as described herein; whereas the same field may not betreated at ineffective times of the growing season or other fields thatare not prone to such attack may go untreated. Such selection steps canbe carried out when practicing each of the methods of use describedherein, i.e., imparting disease resistance to plants, enhancing plantgrowth, effecting pest control (including insects and nematodes),imparting biotic or abiotic stress tolerance to plants, and/ormodulating plant biochemical signaling.

As an alternative to applying an isolated peptide or a compositioncontaining the same to plants or plant seeds in order to impart diseaseresistance in plants, to effect plant growth, to control insects, toimpart stress resistance and/or modulated biochemical signaling to theplants or plants grown from the seeds, transgenic plants or plant seedscan be utilized. When utilizing transgenic plants, this involvesproviding a transgenic plant transformed with a DNA molecule encoding apeptide of the invention and growing the plant under conditionseffective to permit that DNA molecule to impart disease resistance toplants, to enhance plant growth, to control insects, to impart toleranceto biotic or abiotic stress, and/or to modulate biochemical signaling.Alternatively, a transgenic plant seed transformed with a DNA moleculeencoding a peptide of the invention can be provided and planted in soil.A plant is then propagated from the planted seed under conditionseffective to permit that DNA molecule to express the peptide and therebyimpart disease resistance to the transgenic plant, to enhance plantgrowth, to control insects, to impart tolerance to biotic or abioticstress, and/or to modulate biochemical signaling. This transgenicapproach can be used in combination with the recombinant inoculant, ortopical application of the isolated peptide or composition.

The present invention further relates to methods of improvingdesiccation resistance for cuttings removed from ornamental plants,post-harvest disease resistance or desiccation resistance to fruit orvegetables harvested from plants, and/or improved longevity of fruit orvegetable ripeness for fruit or vegetables harvested from plants. Thesemethods involve applying an effective amount of an isolated peptide ofthe present invention or a composition according to the presentinvention to a plant or the locus where the plant is growing. As aconsequence of such application, the peptide contacts cells of the plantor plant seed, and induces desiccation resistance for cuttings removedfrom ornamental plants, post-harvest disease resistance or desiccationresistance to fruit or vegetables harvested from plants, and/or improvedlongevity of fruit or vegetable ripeness for fruit or vegetablesharvested from plants. Alternatively, an effective amount of an isolatedpeptide of the present invention or a composition according to thepresent invention can be applied to a harvested fruit or vegetable. As aconsequence of such application, the peptide contacts cells of theharvested fruit or vegetable, and induces post-harvest diseaseresistance or desiccation resistance to the treated fruit or vegetables,and/or improved longevity of fruit or vegetable ripeness for the treatedfruit or vegetables.

As an alternative to applying an isolated peptide or a compositioncontaining the same to plants or plant seeds in order to inducedesiccation resistance to cuttings removed from ornamental plants,post-harvest disease resistance or desiccation resistance to fruit orvegetables harvested from plants, and/or improved longevity of fruit orvegetable ripeness for fruit or vegetables harvested from plants,transgenic plants or plant seeds can be utilized. When utilizingtransgenic plants, this involves providing a transgenic planttransformed with a DNA molecule encoding a peptide of the invention andgrowing the plant under conditions effective to permit that DNA moleculeto induce desiccation resistance for cuttings removed from ornamentalplants, post-harvest disease resistance or desiccation resistance tofruit or vegetables harvested from the transgenic plants, and/orimproved longevity of fruit or vegetable ripeness for fruit orvegetables harvested from the transgenic plants. Alternatively, atransgenic plant seed transformed with a DNA molecule encoding a peptideof the invention can be provided and planted in soil. A plant is thenpropagated from the planted seed under conditions effective to permitthat DNA molecule to express the peptide and thereby induce desiccationresistance for cuttings removed from ornamental plants, post-harvestdisease resistance or desiccation resistance to fruit or vegetablesharvested from the transgenic plants, and/or improved longevity of fruitor vegetable ripeness for fruit or vegetables harvested from thetransgenic plants.

In these embodiments, it is also possible to select transgenic plants orplant seeds for carrying out the present invention. For example, forfields known to contain a high nematode content, the transgenic plantsor plant seeds can be selectively grown in such fields; whereasnon-transgenic plants or plant seeds can be grown in fields containinglow nematode content. Similarly, for fields having reduced irrigation,the transgenic plants or plant seeds can be selectively grown in suchfields; whereas non-transgenic plants or plant seeds can be grown infields having adequate irrigation. Likewise, for fields prone toflooding, the transgenic plants or plant seeds can be grown in suchfields; whereas non-transgenic plants or plant seeds can be grown infields that are not prone to flooding. As yet another example of suchselection, for fields prone to insect attack at certain times of thegrowing season, the transgenic plants or plant seeds can be selectivelygrown in such fields; whereas non-transgenic plants or plant seeds canbe grown in fields that are not prone to such insect attack. Suchselection steps can be carried out when practicing each of the methodsof use described herein, i.e., imparting disease resistance to plants,enhancing plant growth, effecting pest control (including insects andnematodes), imparting biotic or abiotic stress tolerance to plants,and/or modulating plant biochemical signaling.

The present invention further relates to methods of improvingdesiccation resistance for cuttings removed from ornamental plants,post-harvest disease resistance or desiccation resistance to fruit orvegetables harvested from plants, and/or improved longevity of fruit orvegetable ripeness for fruit or vegetables harvested from plants. Thesemethods involve applying an effective amount of an isolated peptide ofthe present invention or a composition according to the presentinvention to a plant or the locus where the plant is growing. As aconsequence of such application, the peptide contacts cells of the plantor plant seed, and induces desiccation resistance for cuttings removedfrom ornamental plants, post-harvest disease resistance or desiccationresistance to fruit or vegetables harvested from plants, and/or improvedlongevity of fruit or vegetable ripeness for fruit or vegetablesharvested from plants. Alternatively, an effective amount of an isolatedpeptide of the present invention or a composition according to thepresent invention can be applied to a harvested fruit or vegetable. As aconsequence of such application, the peptide contacts cells of theharvested fruit or vegetable, and induces post-harvest diseaseresistance or desiccation resistance to the treated fruit or vegetables,and/or improved longevity of fruit or vegetable ripeness for the treatedfruit or vegetables.

In these embodiments, it is also possible to select plants, cuttings,fruits, vegetables, or the locus to which the isolated peptide orcomposition of the invention is applied. For example, for harvestedcuttings or fruit or vegetables that are being shipped great distancesor stored for long periods of time, then these can be selectivelytreated by applying the isolated peptide or composition of the inventionas described herein; whereas harvested cuttings or fruit or vegetablesthat are being shipped locally and intended to be consumed withoutsubstantially periods of storage can be excluded from such treatment.

As an alternative to applying an isolated peptide or a compositioncontaining the same to plants or plant seeds in order to inducedesiccation resistance to cuttings removed from ornamental plants,post-harvest disease resistance or desiccation resistance to fruit orvegetables harvested from plants, and/or improved longevity of fruit orvegetable ripeness for fruit or vegetables harvested from plants,transgenic plants or plant seeds can be utilized. When utilizingtransgenic plants, this involves providing a transgenic planttransformed with a DNA molecule encoding a peptide of the invention andgrowing the plant under conditions effective to permit that DNA moleculeto induce desiccation resistance for cuttings removed from ornamentalplants, post-harvest disease resistance or desiccation resistance tofruit or vegetables harvested from the transgenic plants, and/orimproved longevity of fruit or vegetable ripeness for fruit orvegetables harvested from the transgenic plants. Alternatively, atransgenic plant seed transformed with a DNA molecule encoding a peptideof the invention can be provided and planted in soil. A plant is thenpropagated from the planted seed under conditions effective to permitthat DNA molecule to express the peptide and thereby induce desiccationresistance for cuttings removed from ornamental plants, post-harvestdisease resistance or desiccation resistance to fruit or vegetablesharvested from the transgenic plants, and/or improved longevity of fruitor vegetable ripeness for fruit or vegetables harvested from thetransgenic plants.

In these embodiments, it is also possible to select transgenic plants orplant seeds for carrying out the present invention. For example,transgenic plants or plant seeds can be selected for growing when it isknown that harvested cuttings or fruit or vegetables are intended to beshipped great distances or stored for long periods of time post-harvest;whereas non-transgenic plants or plant seeds can be selected for growingwhen it is known that harvested cuttings or fruit or vegetables areintended to be shipped locally and/or consumed without substantiallyperiods of storage.

Suitable plants include dicots and monocots, including agricultural,silvicultural, ornamental and horticultural plants, whether in a naturalor genetically modified form. Exemplary plants include, withoutlimitation, alfalfa, apple, apricot, asparagus, avocados, bananas,barley, beans, beech (Fagus spec.), begonia, birch, blackberry,blueberry, cabbage, camphor, canola, carrot, castor oil plant, cherry,cinnamon, citrus, cocoa bean, coffee, corn, cotton, cucumber, cucurbit,eucalyptus, fir, flax, fodder beet, fuchsia, garlic, geranium, grapes,ground nut, hemp, hop, juneberry, juncea (Brassica juncea), jute,lentil, lettuce, linseed, melon, mustard, nectarine, oak, oats, oilpalm, oil-seed rape, olive, onion, paprika, pea, peach, pear,pelargonium, peppers, petunia, pine (Pinus spec.), plum, poplar (Populusspec.), pome fruit, potato, rape, raspberry, rice, rubber tree, rye,sorghum, soybean, spinach, spruce, squash, strawberry, sugar beet, sugarcane, sunflower, tea, teak, tobacco, tomato, triticale, turf,watermelon, wheat and willow (Salix spec.), Arabidopsis thaliana,Saintpaulia, poinsettia, chrysanthemum, carnation, and zinnia.

With respect to modified biochemical signaling, this includes bothenhancement of certain plant biochemical pathways and diminishment ofcertain other plant biochemical pathways. Biochemical signaling pathwaysthat can be altered in accordance with the present invention includegene expression and protein production, production of metabolites, andproduction of signaling molecules/secondary metabolites. Exemplarybiochemical signaling pathways and their modifications include, withoutlimitation, induction of nitric oxide production, peroxide production,and other secondary metabolites; agonist of the ethylene signalingpathway and induction of ethylene-responsive gene expression (see Donget al., Plant Phys. 136:3628-3638 (2004); Li et al., Planta 239:831-46(2014); Chang et al., PLoS One 10,e0125498 (2015), each of which ishereby incorporated by reference in its entirety); agonist of thesalicylic acid signaling pathway and induction of salicylicacid-responsive gene expression (see Dong et al., Plant J. 20:207-215(1999), which is hereby incorporated by reference in its entirety);agonist of the abscisic acid pathway and induction of abscisicacid-responsive gene expression (see Dong et al., Planta 221: 313-327(2005), which is hereby incorporated by reference in its entirety);agonist of the gibberellin signaling pathway and induction ofgibberellin-responsive gene expression (see Li et al., Planta 239:831-46(2014), which is hereby incorporated by reference in its entirety);antagonist of jasmonic acid signaling and inhibiting expression ofjasmonic acid-responsive genes (see Dong et al., Plant Phys.136:3628-3638 (2004), which is hereby incorporated by reference in itsentirety); inducing protease inhibitor expression (see Laluk andMengiste, Plant J. 68:480-494 (2011); Xia et al., Chin. Sci. Bull 56:2351-2358 (2011), each of which is hereby incorporated by reference inits entirety); inducing reactive oxygen species production in planttissues; inducing immune-related and antimicrobial peptide production,such as, without limitation, peroxidase, superoxide dismutase,chitinase, and β-1,3-glucanase (Wang et al., J. Agric. Food Chem.59:12527-12533 (2011), which is hereby incorporated by reference in itsentirety); and inducing expansin gene expression and production (see Liet al., Planta 239:831-46 (2014), which is hereby incorporated byreference in its entirety).

With respect to disease resistance, absolute immunity against infectionmay not be conferred, but the severity of the disease is reduced andsymptom development is delayed. Lesion number, lesion size, and extentof sporulation of fungal pathogens are all decreased. This method ofimparting disease resistance has the potential for treating previouslyuntreatable diseases, treating diseases systemically which might not betreated separately due to cost, and avoiding the use of infectiousagents or environmentally harmful materials.

The method of imparting pathogen resistance to plants in accordance withthe present invention is useful in imparting resistance to a widevariety of pathogens including viruses, bacteria, and fungi. Resistance,inter alia, to the following viruses can be achieved by the method ofthe present invention: Tobacco mosaic virus and Tomato mosaic virus.Resistance, inter alia, to the following bacteria can also be impartedto plants in accordance with present invention: pathogenic Pseudomonasspp., pathogenic Erwinia spp., pathogenic Xanthomonas spp., andpathogenic Ralstonia spp. Plants can be made resistant, inter alia, tothe following fungi by use of the method of the present invention:Fusarium spp. and Phytophthora spp.

With regard to the use of the peptides or compositions of the presentinvention to enhance plant growth, various forms of plant growthenhancement or promotion can be achieved. This can occur as early aswhen plant growth begins from seeds or later in the life of a plant. Forexample, plant growth according to the present invention encompassesgreater yield, increased plant vigor, increased vigor of seedlings(i.e., post-germination), increased plant weight, increased biomass,increased number of flowers per plant, higher grain and/or fruit yield,increased quantity of seeds produced, increased percentage of seedsgerminated, increased speed of germination, increased plant size,decreased plant height (for wheat), greater biomass, more and biggerfruit, earlier fruit coloration, earlier bud, fruit and plantmaturation, more tillers or side shoots, larger leaves, delayed leafsenescence, increased shoot growth, increased root growth, alteredroot/shoot allocation, increased protein content, increased oil content,increased carbohydrate content, increased pigment content, increasedchlorophyll content, increased total photosynthesis, increasedphotosynthesis efficiency, reduced respiration (lower O₂ usage),compensation for yield-reducing treatments, increased durability ofstems (and resistance to stem lodging), increased durability of roots(and resistance to root lodging), better plant growth in low lightconditions, and combinations thereof. As a result, the present inventionprovides significant economic benefit to growers. For example, earlygermination and early maturation permit crops to be grown in areas whereshort growing seasons would otherwise preclude their growth in thatlocale. Increased percentage of seed germination results in improvedcrop stands and more efficient seed use. Greater yield, increased size,and enhanced biomass production allow greater revenue generation from agiven plot of land.

With regard to the use of the peptides or compositions of the presentinvention to control pests (including but not limited to insects andnematodes, which are biotic stressors), such pest control encompassespreventing pests from contacting plants to which the peptide orcomposition of the invention has been applied, preventing direct damageto plants by feeding injury, causing pests to depart from such plants,killing pests proximate to such plants, interfering with insect larvalfeeding on such plants, preventing pests from colonizing host plants,preventing colonizing insects from releasing phytotoxins, interferingwith egg deposition on host plants, etc. The present invention alsoprevents subsequent disease damage to plants resulting from pestinfection.

The present invention is effective against a wide variety of insects(biotic stressors). European corn borer is a major pest of corn (dentand sweet corn) but also feeds on over 200 plant species includinggreen, wax, and lima beans and edible soybeans, peppers, potato, andtomato plus many weed species. Additional insect larval feeding pestswhich damage a wide variety of vegetable crops include the following:beet armyworm, cabbage looper, corn ear worm, fall armyworm, diamondbackmoth, cabbage root maggot, onion maggot, seed corn maggot, pickleworm(melonworm), pepper maggot, and tomato pinworm. Collectively, this groupof insect pests represents the most economically important group ofpests for vegetable production worldwide. The present invention is alsoeffective against nematodes, another class of economically importantbiotic stressors. Soybean Cyst Nematode (Heterodera glycines) is a majorpest of soybeans. Reniform Nematode (Rotylenchulus reniformis) is amajor pest of cotton as can parasitize additional crop species, notablysoy and corn. Additional nematode pests include the root knot nematodesof the genus Meloidogyne (particularly in cotton, wheat, and barley),cereal cyst nematodes of the genus Heterodera (particularly in soy,wheat, and barley), root lesion nematodes of the genus Pratylenchus,seed gall nematodes of the genus Anguina (particularly in wheat, barley,and rye), and stem nematodes of the genus Ditylenchus. Other bioticstressors include arachnids, weeds, and combinations thereof.

With regard to the use of the peptides or compositions of the presentinvention to impart abiotic stress resistance to plants, such abioticstress encompasses any environmental factor having an adverse effect onplant physiology and development. Examples of such environmental stressinclude climate-related stress (e.g., drought, flood, frost, coldtemperature, high temperature, excessive light, and insufficient light),air pollution stress (e.g., carbon dioxide, carbon monoxide, sulfurdioxide, NO_(R), hydrocarbons, ozone, ultraviolet radiation, acidicrain), chemical (e.g., insecticides, fungicides, herbicides, heavymetals), nutritional stress (e.g., over- or under-abundance offertilizer, micronutrients, macronutrients, particularly potassium,nitrogen derivatives, and phosphorus derivatives), and improved healingresponse to wounding. Use of peptides of the present invention impartsresistance to plants against such forms of environmental stress.

A further aspect of the present invention relates to the use of thepeptides of the present invention as a safener in combination with oneor more of the active agents (i.e., in a composition or in separatecompositions) for the control of aquatic weeds in a body of water asdescribed in U.S. Publ. No. 20150218099 to Mann, which is herebyincorporated by reference in its entirety.

Yet another aspect of the present invention relates to the use of thepeptides of the present invention as a plant strengthener in acomposition for application to plants grown under conditions of reducedwater irrigation, which composition also includes at least oneantioxidant and at least one radiation manager, and optionally at leastone plant growth regulator, as described in U.S. Publ. No. 20130116119to Rees et al., which is hereby incorporated by reference in itsentirety.

The methods of the present invention involving application of thepeptide or composition can be carried out through a variety ofprocedures when all or part of the plant is treated, including leaves,stems, roots, propagules (e.g., cuttings), fruit, etc. This may (butneed not) involve infiltration of the peptide into the plant. Suitableapplication methods include high or low pressure spraying, injection,and leaf abrasion proximate to when peptide application takes place.When treating plant seeds, in accordance with the application embodimentof the present invention, the hypersensitive response elicitor proteinor polypeptide can be applied by low or high pressure spraying, coating,immersion (e.g., soaking), or injection. Other suitable applicationprocedures can be envisioned by those skilled in the art provided theyare able to effect contact of the hypersensitive response elicitorpolypeptide or protein with cells of the plant or plant seed. Oncetreated with the peptides or compositions of the present invention, theseeds can be planted in natural or artificial soil and cultivated usingconventional procedures to produce plants. After plants have beenpropagated from seeds treated in accordance with the present invention,the plants may be treated with one or more applications of the peptidesor compositions of the invention to impart disease resistance to plants,to enhance plant growth, to control insects on the plants, to impartbiotic or abiotic stress tolerance, to improve desiccation resistance ofremoved cuttings, to impart post-harvest disease resistance ordesiccation resistance to harvested fruit or vegetables, and/or improvedlongevity of fruit or vegetable ripeness for harvested fruit orvegetables.

Where the peptides are applied in the form of a recombinant beneficialmicrobe, these microbes can be applied in the form of an aqueoussolution comprising a suspension of such beneficial microbes, which isthen applied to the plant by spraying, coating, or immersion asdescribed above. When treating plant seeds, in accordance with theapplication embodiment of the present invention, the microbes can beapplied by low or high pressure spraying, coating, immersion (e.g.,soaking), or injection. Other suitable application procedures can beenvisioned by those skilled in the art provided they are able to effectcontact of the beneficial microbes with cells of the plant or plantseed. In accordance with the application embodiment of the presentinvention, the beneficial microbes can be applied to plants or plantseeds in dry form. By way of example, dry application of microbes can beaccomplished using bacterial or fungal products such as Kodiak® HB,available from Chemtura, and T-22™ HC, available from BioWorks. Oncetreated with the microbes of the present invention, the seeds can beplanted in natural or artificial soil and cultivated using conventionalprocedures to produce plants. After plants have been propagated fromseeds treated in accordance with the present invention, the plants maybe treated with one or more applications of the microbes of theinvention or the peptides, fusion proteins, or compositions of theinvention, to impart disease resistance to plants, to enhance plantgrowth, to control insects on the plants, to impart biotic or abioticstress tolerance, to improve desiccation resistance of removed cuttings,to impart post-harvest disease resistance or desiccation resistance toharvested fruit or vegetables, and/or improved longevity of fruit orvegetable ripeness for harvested fruit or vegetables.

The peptides or compositions of the invention can be applied to plantsor plant seeds in accordance with the present invention alone or in amixture with other materials. Alternatively, the peptides orcompositions can be applied separately to plants with other materialsbeing applied at different times.

In the alternative embodiment of the present invention involving the useof transgenic plants and transgenic seeds, a peptide of the inventionneed not be applied topically to the plants or seeds. Instead,transgenic plants transformed with a DNA molecule encoding a peptide ofthe invention are produced according to procedures well known in theart. A vector suitable for expression in plants (i.e., containingtranslation and transcription control sequences operable in plants) canbe microinjected directly into plant cells by use of micropipettes totransfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics,202:179-85 (1985), which is hereby incorporated by reference in itsentirety. The genetic material may also be transferred into the plantcell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982),which is hereby incorporated by reference in its entirety.

Another approach to transforming plant cells with a gene encoding thepeptide of the invention is particle bombardment (also known asbiolistic transformation) of the host cell. This can be accomplished inone of several ways. The first involves propelling inert or biologicallyactive particles at cells. This technique is disclosed in U.S. Pat. Nos.4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which arehereby incorporated by reference. Generally, this procedure involvespropelling inert or biologically active particles at the cells underconditions effective to penetrate the outer surface of the cell and tobe incorporated within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the heterologous DNA.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried bacterial cells containingthe vector and heterologous DNA) can also be propelled into plant cells.

Yet another method of introduction is fusion of protoplasts with otherentities, either minicells, cells, lysosomes or other fusiblelipid-surfaced bodies. Fraley, et al., Proc. Natl. Acad. Sci. USA,79:1859-63 (1982), which is hereby incorporated by reference in itsentirety. The DNA molecule may also be introduced into the plant cellsby electroporation. Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824(1985), which is hereby incorporated by reference in its entirety. Inthis technique, plant protoplasts are electroporated in the presence ofplasmids containing the expression cassette. Electrical impulses of highfield strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and regenerate.

Another method of introducing the DNA molecule into plant cells is toinfect a plant cell with Agrobacterium tumefaciens or A. rhizogenespreviously transformed with the gene. Under appropriate conditions knownin the art, the transformed plant cells are grown to form shoots orroots, and develop further into plants. Generally, this procedureinvolves inoculating the plant tissue with a suspension of bacteria andincubating the tissue for 48 to 72 hours on regeneration medium withoutantibiotics at 25-28° C. Agrobacterium is a representative genus of thegram-negative family Rhizobiaceae. Its species are responsible for crowngall (A. tumefaciens) and hairy root disease (A. rhizogenes). The plantcells in crown gall tumors and hairy roots are induced to produce aminoacid derivatives known as opines, which are catabolized only by thebacteria. The bacterial genes responsible for expression of opines are aconvenient source of control elements for chimeric expression cassettes.In addition, assaying for the presence of opines can be used to identifytransformed tissue. Heterologous genetic sequences can be introducedinto appropriate plant cells, by means of the Ti plasmid of A.tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid istransmitted to plant cells on infection by Agrobacterium and is stablyintegrated into the plant genome. J. Schell, Science, 237:1176-83(1987), which is hereby incorporated by reference in its entirety.

After transformation, the transformed plant cells must be regenerated.Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co.,New York, 1983); and Nasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. 1, 1984, and Vol. Ill(1986), which are hereby incorporated by reference in their entirety.

It is known that practically all plants can be regenerated from culturedcells or tissues. Means for regeneration varies from species to speciesof plants, but generally a suspension of transformed protoplasts or apetri plate containing transformed explants is first provided. Callustissue is formed and shoots may be induced from callus and subsequentlyrooted. Alternatively, embryo formation can be induced in the callustissue. These embryos germinate as natural embryos to form plants. Theculture media will generally contain various amino acids and hormones,such as auxin and cytokinins. It is also advantageous to add glutamicacid and proline to the medium, especially for such species as corn andalfalfa. Efficient regeneration will depend on the medium, on thegenotype, and on the history of the culture. If these three variablesare controlled, then regeneration is usually reproducible andrepeatable.

After the expression cassette is stably incorporated in transgenicplants, it can be transferred to other plants by sexual crossing. Any ofa number of standard breeding techniques can be used, depending upon thespecies to be crossed.

Once transgenic plants of this type are produced, the plants themselvescan be cultivated in accordance with conventional procedure with thepresence of the gene encoding the hypersensitive response elicitorresulting in disease resistance, enhanced plant growth, control ofinsects on the plant, abiotic or biotic stress tolerance, improveddesiccation resistance of removed cuttings, post-harvest diseaseresistance or desiccation resistance in harvested fruit or vegetables,and/or improved longevity of fruit or vegetable ripeness for harvestedfruit or vegetables.

Alternatively, transgenic seeds are recovered from the transgenicplants. These seeds can then be planted in the soil and cultivated usingconventional procedures to produce transgenic plants. The transgenicplants are propagated from the planted transgenic seeds under conditionseffective to impart disease resistance to plants, to enhance plantgrowth, to control insects, to impart abiotic or biotic stresstolerance, to improve desiccation resistance of removed cuttings, toimpart post-harvest disease resistance or desiccation resistance inharvested fruit or vegetables, and/or to impart improved longevity offruit or vegetable ripeness for harvested fruit or vegetables.

When transgenic plants and plant seeds are used in accordance with thepresent invention, they additionally can be treated with the samematerials as are used to treat the plants and seeds to which a peptideof the invention or composition of the invention is applied. These othermaterials, including peptides or composition of the invention, can beapplied to the transgenic plants and plant seeds by the above-notedprocedures, including high or low pressure spraying, injection, coating,and immersion. Similarly, after plants have been propagated from thetransgenic plant seeds, the plants may be treated with one or moreapplications of the peptides or compositions of the invention to impartdisease resistance, enhance growth, control insects, abiotic or bioticstress tolerance, desiccation resistance of removed cuttings,post-harvest disease resistance or desiccation resistance in harvestedfruit or vegetables, and/or improved longevity of fruit or vegetableripeness for harvested fruit or vegetables.

Such transgenic plants may also be treated with conventional planttreatment agents, e.g., bacteriocidal or biocidal agents, proteaseinhibitors, non-ionic surfactants, fertilizers, herbicides,insecticides, fungicides, nematicides, biological inoculants, plantregulators, and mixtures thereof, as described above.

Examples

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Example 1—Determination of a Minimal HR-Eliciting Sequence

The minimal eliciting sequence derived from was developed based on thesequence of the hrex gene from Xanthomonas campestris pv. pelargonii. Anumber of truncated and mutated sequences were synthesized and testedfor Hypersensitive Response induction (results in Table 4). HR intobacco was tested as described in Wei, Science 257:85-88 (1992), whichis hereby incorporated by reference in its entirety. Briefly, peptideswere dissolved at a concentration of 500 μg/ml in aqueous solution. Fourserial dilutions were performed with an equal volume of water, yieldingpeptide samples at 500, 250, 125, 62.5, 31.25 μg/ml peptide solutions.Nicotiana tabacum cultivar xanthi plants were used at 5-7 weeks old(preflowering). Leaves were lightly punctured with a toothpick in amiddle leaf panel. Peptide solutions were then infused via needle-lesssyringe into the wound, filling the panel. Each peptide sample wasinfused into a leaf of 2 different plants. The leaves were observed andscored over the next 48 hours for withering and browning, lesionstypical of programmed cell death. These studies had three main goals:(1) determine the minimal sequence sufficient for HR elicitation (2)increase the solution stability of the peptides; (3) make disruptivemutations to verify the residues which are most important for HRelicitation; (4) make conservative mutations to identify the degree ofspecificity for particular amino acids.

The wild-type sequence from Xanthomonas campestris pv. pelargoniicontains several methionine residues, which are prone to oxidation.Mutant peptides (P5-2, and P5-3) contain Met to Ala mutations, but thesemutations do not affect the elicitation of HR, suggesting that themethionine residues are dispensable for HR activation.

Based on the P5 and P5a sequences (SEQ ID NOS: 8 and 9), disruptive orconservative single mutations were introduced at specific residueswithin the sequence. In the case of Leucine residues, these were mutatedto glutamic acid (disruptive due to the introduction of a negativecharge) or valine (conservative). The intervening sequences, dependingon the identity of the amino acid in question, were mutated to have anegative charge (aspartic acid or glutamic acid), have a hydrophobicsidechain (valine), a minimal sidechain (alanine), or a small polarsidechain (serine). These mutant peptides were tested for elicitation ofthe hypersensitive response. Additional mutations are chosen based onthe initial HR results. For those amino acids that were important for HRelicitation, more conservative mutations were chosen to determine thespecificity of interactions. The leucine residues were mutated toisoleucine, valine, phenylalanine or tyrosine, with the latter 2residues being less conservative. As above, these mutants were testedfor HR elicitation.

TABLE 4 Peptide SEQ name Sequence ID NO: HR: P5 SAGSEQQLDLLLMFIMMMLQQ  8 + P5a SAGSEQQLDQLLLMFIMMMLQQ   9 + P5-2 SAGSEQQLDLLLMFIAAALQQ  10 +P5-3 SAGSEQQLDLLLAFIAAALQQ  11 + P5-4 SAGSEQQLELLLAFIAAALQQ  12 + P5-5QLELLLAFIAAALQQ 139 + P5-6 SAGSEQQLDLLLAFIAAAL 140 - P5-7SEEEEELDLLLAFIAAAL  13 - P5-8 SEEEEELDLLLAFIAAALQQ  14 Weak+ P5-9LDLLLAFIAAALEEEEEEE  15 - P5-10 LDLLLAFIEEELEEEE  16 - P5-11SEEELDLLLAFIAAALEE  17 - P5-12 SEEELDLLLAFIEEELEE  18 - P5-13SEEELDLLLAFIAAALDD  19 - P5-14 SEEEEELDLLLAFIAAALGG  20 Weak+ P5-15SEEEEELDLLLAFIAAALQ  25 - P5-16 SEEEEELDLLLAFIAAALS  26 - P5-17SEEEEELDLLLAFIAAALA  27 - P5-18 SEEEEELDLLLAFIAAALE  28 - P5-19SELELLLAFIAAALEEEEE  29 + P5-20 SELELLLEFIEEELEE  36 - P5-21SEEQLELLLAFIAAALQQEE  33 + P5-22 SEEELELLLAFIAAALEEEE  30 + P5-23SEEEEELDQLLLAFIAAALQQ  50 Weak+ P5-24 SEEEEELDQLLLAFIAAAL  51 - P5-25SEEEEQLDQLLLAFIAAALQQ  52 - P5-26 SEEQLDLLLAFIAAALQEE  34 + P5-27SEEQLDQLLLAFIAAALQEE  53 - P5-28 SEEQLDQLLLAFIAAALEE  54 Weak+ P5-29SEEELDLLLMFIMMMLEE  42 + P5-30 SEEELDQLLLMFIMMMLEE  55 + P5-31SEEEQLDLLLMFIMMMLEE  43 + P5-32 SEEEQLDQLLLMFIMMMLEE  56 + P5-33SEEEQLDLLLMFIMMMLQEE  44 + P5-34 SEEEQLDQLLLMFIMMMLQEE  57 + P5-35SAGSEQQEDLLLMFIMMMLQQ  71 Weak+ P5-36 SAGSEQQLDELLMFIMMMLQQ  72 + P5-37SAGSEQQLDLELMFIMMMLQQ  73 - P5-38 SAGSEQQLDLLEMFIMMMLQQ  74 - P5-39SAGSEQQLDLLLEFIMMMLQQ  75 Strong+ P5-40 SAGSEQQLDLLLMEIMMMLQQ  76Strong+ P5-41 SAGSEQQLDLLLMFEMMMLQQ  77 - P5-42 SAGSEQQLDLLLMFIEMMLQQ 78 Weak+ P5-43 SAGSEQQLDLLLMFIMEMLQQ  79 Strong+ P5-44SAGSEQQLDLLLMFIMMELQQ  80 Weak+ P5-45 SAGSEQQLDLLLMFIMMMEQQ  81 - P5-46SEEQLDQLLLMFIMMMLQQEE  58 + P5-47 SEEQLDLLLMFIMMMLQQEE  45 + P5-48SEEQLDLLLEFIEEELQQEE  39 - P5-49 SAGSEQQEDLLLAFIAAALQQ 510 - P5-50SAGSEQQLDELLAFIAAALQQ 511 Weak+ P5-51 SAGSEQQEDELLAFIAAALQQ 512 - P5-52SAGSEQQEDELLMFIMMMLQQ 513 - P5-53 SAGSEQQEDQLLLMFIMMMLQQ  98 - P5-54SAGSEQQLDQELLMFIMMMLQQ  99 - P5-55 SAGSEQQLDQLLLAFIAAALQQ 130 - P5-56SEEQEELLLAFIAAALQQEE 514 - P5-57 SEEQLEELLAFIAAALQQEE 515 + P5-58SEEQEEELLAFIAAALQQEE 516 - P5-61 SAGSEQQEDLLLAFIALQQ 519 - P5-62SAGSEQQLDELLAFIALQQ 520 - P5-63 SAGSEQQLDLLLEFIALQQ 521 - P5-64SAGSEQQLDLLLAEIALQQ 522 - P5-65 SAGSEQQLDLLLAFIEALQQ 523 - P5-66SAGSEQQLDLLLAFIAEALQQ 524 Weak+ P5-67 SAGSEQQLDLLLAFIEAALQQ 525 - P5-68SAGSEQQLDLLLAFIAAELQQ 526 - P5-69 SAGSEQQLDLLLAEIAAALQQ 527 - P5-70SAGSEQQLDLLLEFIAAALQQ 528 - P5-71 SAGSEQQEDLLLAFIAAALQQ 529 - P5-72SAGSEQQLDELLAFIAAALQQ 530 - P5-73 SQAGSEQLDLLLMFIMMMLQQ 531 + P5-74AEQGSSQLDLLLMFIMMMLQQ 532 + P5-75 NQGISEKQQLDLLLMFIMMMLQQ 533 Strong+P5-76 NQGISEKQQLDLLLAFIAAALQQ 534 Weak+ P5-77 NFGTPDSTVQNPQDASKPNQLDL535 Weak+ LLMFIMMMLQQ P5-78 NFGTPDSTVQNPQDASKPNQLDL 536 Weak+LLAFIAAALQQ P5-79 ITPDGQGGGQIGDNPQLDLLLMF 537 Strong+ IMMMLQQ P5-80ITPDGQGGGQIGDNPQLDLLLAF 538 Weak+ IAAALQQ P5-87 SEEQLDLLLAFIAAALQQEE539 - P5-88 SEEQLELLLAFIAAALQEE 540 +

Example 2—Stability Tests of Variant Peptides

Peptides were assessed for one or more of solubility, stability againstchemical degradation, effect of bulking agents on solution stability,oxidation protection and solution stability studies.

Stability against chemical degradation was assessed in various pHbuffers by creating 0.2% AI solutions of pure, chemically synthesizedpeptide in deionized water, 0.25% weight to volume of Proxel® GXL(biocide), and 50 millimolar (mM) of nine buffers (separately) asfollows: Citrate, pH 5.6, MES pH 6.0, MOPS pH 6.5, Citrate pH 7.2, EDDSpH 7.3, imidazole pH 7.5, EDTA pH 8, Phosphate pH 8, and TES pH 8. Thesolutions were observed on HPLC for evidence of degradation (% loss ofthe peptide signal over time, relative to the time 0 sample) over aperiod of weeks at elevated temperature (50° C.). Precipitation of P5and P5-5 was noted in several samples, particularly those with pH<7.Other peptides, notably P5-9 and P5-21 remained in solution. Thesecorrelate with the lower hydrophobicity values for P5-9.

Peptide P5 exhibited poor solubility below pH 7.0, which distorted theexperimental results (not shown). For the buffer solutions above pH 7.0,generally poor stability was observed, with the best results in an EDTAbuffer at pH 8.0 (about 40% remaining after 14 days (FIG. 1). P5-9contains a C-terminal glutamate sequence for solubility enhancement andmutations of methionine residues for increased resistance to oxidation.This peptide exhibited better solubility performance at 500 ug/ml aswell as better stability compared with P5 (FIG. 2). Citrate at pH 7.2and Phosphate at pH 8.0 exhibited above 80% remaining after 14 days.Notably, after 45 days, 81% of the original peptide remained for thecitrate pH 7.2 sample and 79% for the phosphate pH 8.0.

P5-21 is a minimal sequence necessary for HR elicitation containing bothN- and C-terminal solubility enhancing sequences and methionine toalanine mutations. It exhibited good solubility at pH 5.6 and higher aswell as excellent stability (>90%) for all buffers pH >7.0 except EDTA(around 80%). Results are shown in FIG. 3.

Samples of material P5, P5-21, and P5-25 bulked with maltodextrin andeither phosphate buffer (pH 8.0) or citrate buffer (pH 7.2) were testedfor stability at room temperature for 48 hours. No significantdegradation (more than 5% loss) was observed.

Solution stability studies were carried out by creating 0.09% AIsolutions of pure, chemically synthesized peptide in deionized water, 50mM of pH buffer, 0.25% Proxel GXL, and 0-50% isopropanol. Peptidessolutions were analyzed by HPLC for % loss of the peptide signal overtime during incubation at 50° C., relative to the time 0 sample.Peptides were analyzed until the remaining peptide concentrationdecreased to 80% of original (20% degradation). Results are summarizedin Table 5 below.

TABLE 5 Formulation Stability of P5 Variants Time before 20% Peptide SEQID NO: Formulation: degradation (days) P5 8 50 mM phosphate pH 8.0 2130% IPA 5 mM DTPA P5-21 33 50 mM citrate pH 7.2 41 20% IPA P5-25 52 50mM citrate pH 7.2 53 P5-27 53 50 mM imidazole pH 7.5 50 50% IPA IPA:isopropanol, DTPA: diethylenetriaminepentaacetic acid

Example 3—Induction of Resistance to Tobacco Mosaic Virus

Peptides were tested for the induction of resistance to tobacco mosaicvirus (TMV) in tobacco. Briefly, three tobacco plants at 6-8 weeks oldwere selected per group (samples and controls). The bottom-most leaf ofthe plant was covered and the plant was sprayed with a solution of water(untreated control—UTC), peptide, or Proact (positive control). Thespray was applied until the leaves were fully wetted, indicated byliquid dripping from the leaves. The plants were then allowed to dry andthe leaf covering was removed.

Three days post-treatment, the previously-covered leaf and a leaf on theopposite side of the plant were then lightly dusted with diatomaceousearth and 20 ul of a 1.7 ug/ml solution of purified tobacco mosaic viruswas applied. The TMV solution was then spread across the leaf surface bylightly rubbing solution and the diatomaceous earth across the surfaceof the leaves. Two minutes after inoculation, the diatomaceous earth wasrinsed off the leaves with water. 3 days after TMV inoculation, theleaves were scored based on the number of TMV lesions observed. The leafwas also scored for signs of the hypersensitive response, includingyellowing and wilting of the affected leaves.

Effectiveness described in Table 6 refers to the % decline in TMVlesions on treated vs UTC plants. A reduction of TMV on covered leavesindicates a systemic immune response in the plant while reduction onuncovered leaves indicates a local response. Asterisks indicate that theP-value derived from a T-test was <0.05.

TABLE 6 Summary of TMV Resistance SEQ ID Concentration EffectivenessEffectiveness Peptide NO: (ug/ml) Uncovered (%) Covered (%) P5 8 10 68*50  P5-2 10 20 25  90* P5-3 11 20 55  70* P5-4 12 20 97* 98* P5-5 139 2084* 97* P5-6 140 20 62  36  P5-7 13 20 71  91* P5-8 14 20 99* 99* P5-2133 20 94* 81* P5-22 30 20 82  62  P5-25 52 20 91* 93* P5-46 58 20 85*74* P5-47 45 20 87* 78*

Example 4—Induction of Nematode Resistance in Soy Plants

The effectiveness of peptide treatment in suppressing the growth ofsoybean cyst nematodes (SCN) was assessed in soy. Soy was planted in a1:1 sand/Turface mixture in a greenhouse (temperature held at 28° C.),with 10 plants per treatment group. 14 days after planting, plants weresprayed with a 2.0 μg/ml solution of peptide or a control solutionwithout peptide. 4,000 freshly harvested SCN eggs were added to theplants 48 hours after peptide application. 30 days after pathogenintroduction, the plants were harvested and the cysts were collected andcounted using an elutriator.

In two separate trials, application of P5 (SEQ ID NO: 8) at 2.0 ug/mlcaused a significant reduction in SCN populations. In trial #1, thecontrol population contained an average of 133.5 cysts per plantcompared with an average of 69.7 cysts per plant for the P5 treatmentgroup (P=0.004). In trial #2, the control population contained anaverage of 104.9 cysts per plant compared with an average of 54.5 cystsper plant for the P5 treatment group (P=0.019). These results suggestthat the peptides of the current invention strongly activate soy plantdefenses against nematode infiltration.

Additional experiments will be performed to examine the effectiveness ofpeptide treatment in suppressing the growth of soybean cyst nematodesusing one or more of the other peptides in Tables 1 and 2, including,without limitation, P5-21 and P5-25. See Example 8 below.

Example 5—Drought Resistance in Corn

The effectiveness of peptide treatment in reducing drought stress wasassessed in corn and soy. 3.5 inch pots were filled with Sunshine #1soil (SunGro Horticulture), fertilized with a 20-10-20 mixture. The soilwas soaked and drained overnight. Seeds (either corn or soy, manuallyinspected to ensure uniform seed size) were planted at a depth of 1 inchfor germination. Plants were grown in a greenhouse under 16 hour lightdays at >70° F. and 8 hour dark nights at >65° F. Prior to droughtconditions, the plants were well-watered.

When the plants reached the V1 stage, plants were culled to achieve auniform height (abnormally large and small plants were removed). Plantswere then randomly assigned to control (spray without peptide) ortreatment (spray with peptide) groups and heights were measured. Peptidewas made up in a solution of 0.2 ug/ml or 2 ug/ml in distilledwater+0.01% Tween-20, and applied as a fine mist from a spray bottleuntil the solution drips from the leaves. After the peptide solutionswere dried, the plants were again randomized in a randomized completeblock design. Drought stress was initiated after the peptide treatment.This was caused by maintaining the water level at 25-50% of the maximumcapacity for water (capacity was decided as the weight of the pot filledwith saturated soil minus the weight of the filled pot prior to addingwater).

The drought test phase ended after 2-3 weeks. At that time, the plantheight was again measured and the growth rate was calculated as thedifference between this and the previously-recorded height. Theabove-ground part portions of the plants were harvested and weighed. Theabove-ground portion was also dried in an oven at 70° C. for 72 hoursand a dry weight was obtained. All calculations were compared withmatched untreated control plants.

The drought testing procedure was carried out in corn using a treatmentof P5 (SEQ ID NO: 8). Treatment with 2.0 ug/ml of peptide caused a 3.09%increase in the growth rate, a 10.04% increase in the dry weight(P<0.05), and a 4.01% increase in fresh weight.

As shown in Table 7, additional peptides caused a drought resistancephenotype.

TABLE 7 Summary of Drought Resistance SEQ ID Concentration Dry WeightFresh Weight Peptide NO: (ug/ml) Increase (%) Increase (%) P5-46 58 2.03.71 9.05** P5-76 534 0.2 5.33* 2.00 P5-35 71 2.0 −6.57* −6.05* *P <0.1, **P < 0.05

Experimental results for P5-21 and P5-25 were not statisticallysignificant. One notable result is P5-35. Although this result is anegative phenotype under treatment, this shows that the peptide has somebioactivity. It has been shown previously with other harpin proteinsthat over-application can result in a negative response. Futureexperiments could show that lower concentration application of thispeptide can cause a positive biological response. See Example 10 below.

Example 6—Root Knot Nematode (RKN) Resistance in Tomato

‘Rutgers’ tomato seedlings were transplanted into pasteurized sandy soilin 5 inch clay pots. The plants were spray-treated with a peptidesolution until the leaves were saturated immediately aftertransplanting. 2 days after transplant, the plants were inoculated withroot knot nematode (Meloidogyne incognita) eggs (5000 eggs per pot).Plants were maintained in a greenhouse for about 60 dayspost-inoculation, corresponding to 2 life cycles for the nematode. Twoadditional spray applications were made 21 and 42 days aftertransplanting.

For these trials, controls treatments included a commercial standard(Vydate) positive control; an untreated, RKN-inoculated control; and anuntreated uninoculated control. At the end of each trial, the followingmeasures were taken: root gall rating classification (based on % ofroots with galling, 1—minimal: <5% roots with galls, 2—slight:5-25%roots with galls, 3—moderate: 26-50% roots with galls, 4—heavy: over 50%of roots with galls) and counting of the number of RKN eggs per gram ofroot.

For spray-treatment with P5 at 46.7 ug/ml, a significant reduction inroot galling was observed (4 in untreated plants vs 2-3 in treatedplants). Likewise, a reduction in egg counts was observed: an average of165,600 for untreated plants vs 52,000 for treated plants, a 69%decrease, p=0.02.

Example 7—Drought Resistance in Soy

The effectiveness of peptide treatment in reducing drought stress wasassessed in soy. 3.5 inch pots were filled with Sunshine #1 soil (SunGroHorticulture), fertilized with a 20-10-20 mixture. The soil was soakedand drained overnight. Seeds (soy, manually inspected to ensure uniformseed size) were planted at a depth of half inch for germination. Plantswere grown in a greenhouse under 16 hour light days at >70° F. and 8hour dark nights at >65° F. Prior to drought conditions, the plants werewell-watered.

When the plants reached the growth stage when first trifoliate expanded,plants were culled to achieve a uniform height (abnormally large andsmall plants were removed). Plants were then randomly assigned tocontrol (spray without peptide) or treatment (spray with peptide) groupsand heights were measured. Peptide was made up in a solution of 0.2ug/ml or 2 ug/ml in distilled water+0.04% Tween-20, and applied as afine mist from a spray bottle until the solution drips from the leaves.After the peptide solutions were dried, the plants were again randomizedin a randomized complete block design. Cyclic drought stress wasinitiated after the peptide treatment. Plants were subjected to at leastthree drought cycles of drought stress (3-5 days of withholding waterand 1 day irrigated with saturating amount of water) before harvesting.

The drought test phase ended after 2-3 weeks. At that time, the plantheight was again measured and the growth rate was calculated as thedifference between this and the previously-recorded height. Theabove-ground part portions of the plants were harvested and weighed toobtain fresh weight. The above-ground portion was also dried in an ovenat 70° C. for 72 hours to obtain dry weight. All calculations werecompared with matched untreated control plants.

Results are shown in Table 8 below. Asterisks indicate statisticalsignificance by P-value (*: P<0.1 and **: P<0.05).

TABLE 8 Summary of Drought Resistance SEQ ID Concentration Dry WeightFresh Weight Peptide NO: (ug/ml) Increase (%) Increase (%) P5-27 53 2.02.04 4.07* P5-33 44 2.0 5.02** 4.10 P5-75 533 0.2 3.73 8.78** P5-75 5332.0 4.37 8.55**

Example 8—Comparison of Peptides with Chemical Nematode Agents in Soy

The effectiveness of peptides was tested in soy (Sheyenne variety) andcompared with current anti-nematode chemicals. Briefly, soy seeds werecommercially coated with peptide at rates of 10, 30, or 90 ug peptideper seed or with chemical nematicides Avicta Complete, Clariva Complete,Poncho/Votivo, or Velum total. 3 soy seeds were planted in a mixture oftopsoil and sand (pasteurized) in a 10 cm diameter pot and wereinoculated with 1500 eggs per pot at planting. 10 replicate pots wereused per experimental condition. After plants emerged from the soil, theplants were thinned to one plant per pot. Plants were harvested about 45days after planting. The following measures were taken: (1) Fresh shootand root weights; (2) Root condition graded 0-10 for the presence ofdisease; (3) Nematode cysts per plant; (4) Nematode eggs per plant; andoptionally (5) Male nematodes. The following values were calculated:eggs per gram of root, cysts per gram of root, and eggs per cyst.

P5 (SEQID: 8) did not exhibit a significant reduction in cysts or eggs,but did cause a ˜30% reduction in the number of male nematodes. However,the chemical treatments caused a greater reduction in the malenematodes.

P5-21 (SEQID: 33) caused a reduction in cysts per gram root: 91% at 10ug, 80% at 30 ug, and 86% at 90 ug. These were numerically greater thanthe results for Clariva Complete (76%) and Avicta Complete (78%). Theresults were statistically significantly greater than those ofPoncho/Votivo (58%). There was also a significant reduction in eggs pergram root: 93% for a bug treatment, 74% for a 30 ug treatment, 92% for a90 ug treatment. These were numerically greater than those of ClarivaComplete (75%) and Avicta Complete (72%) for all tested rates. Theresults for P5-21 at bug and 90 ug were statistically significantlygreater than the results for Poncho/Votivo (49%).

P5-25 (SEQID: 52) also caused a strong reduction in cysts per gram ofroot: 92% at 10 ug, 82% at 30 ug, and 91% at 90 ug. These werenumerically greater than the results for Clariva Complete (76%) andAvicta Complete (78%). The results at bug and 90 ug were statisticallysignificantly greater than Poncho/Votivo (58%). Likewise, there was asignificant reduction in the eggs per gram root: 94% at 10 ug, 85% at 30ug, and 93% at 90 ug. These results were numerically better than ClarivaComplete (75%) and Avicta Complete (72%). The results were alsostatistically significantly greater than those of Poncho/Votivo (49%).

Example 9—Stimulation by P5 of Anti-Nematode Root Secretions in Soy

Soybean seeds coated with P5 (SEQ ID NO: 8) or a mock treatment werewetted and germinated in brown paper for 48 hours. Seedlings were thenplaced in a beaker with 3 ml distilled water per seedling for 24 hours.The liquid exudate was then collected and 1 ml was added to each well ofa 24-well plate. 300 soybean cyst nematode Heterodera glycines eggs wereadded into a plastic micro-sieve mesh and placed on each of the 24wells. After incubating for 3 days, the number of hatched nematodes atthe well bottom was counted. In the mock-treated exudate wells, countsrevealed an average of 40 hatched nematodes. In the p5-treated exudatewells, an average of 30 hatched nematodes was counted. This was astatistically significant difference (P<0.05).

Example 10—Corn Drought Performance in the Field

Peptide P5 (SEQ ID: 8) was tested for its efficacy in reducing droughtstress under field conditions. Briefly, a site was chosen in Hughson,Calif., where rainfall is minimal and irrigation is required forgrowing. Corn hybrid seeds 639STX (Heine Seed Company) were coated withP5 at 12, 35, or 105 ug peptide per seed or a mock treatment. Theplanting field was divided 3 row (30″ spacing)×25 foot plots. Corn wasplanted at a rate of 32,000 viable seeds per acre (6.5 inches betweenseeds on 30 inch rows. Four border rows (10 ft) were planted on allsides of the trial to mitigate environmental effects. Each treatmentgroup was replicated 4 times and arrayed in a Randomized Complete Blockdesign.

Irrigation was managed in the following way: The site was initiallyirrigated to assure a good plant stand and early growth. Two droughtperiods were initiated. The first started at the V-3 plant growth stageand ended when leaf curling was visible in the plants. The seconddrought period started at tassel emergence and ended at the end ofpollen shed. During both drought periods, the plants were monitored toavoid reaching a permanent wilting point.

Treatment with P5 resulted in an increase of 20.2 bushels per acre(Bu/Ac) for 12 ug/seed treatment, an increase of 11.4 Bu/Ac for 35ug/seed treatment, and an increase of 19.7 Bu/Ac for a 105 ug/seedtreatment rate. These results compared with an average yield of 123.6Bu/Ac for untreated control. The growers also observed an increase inmetrics associated with the size of the corn ear. The number of rows percorn ear increased from 12.3 (UTC) to 12.7 for 12 ug/seed, remainedunchanged for 35 ug/seed, and increased to 13.1 for 105 ug/seed. Themass of the corn ears also increased from 90.1 grams per ear (UTC) to104.8 g for 12 ug/seed, 98.4 g for 35 ug/seed, and 104.5 g for 105ug/seed. The number of kernels per row was 25.9 for UTC. This increasedto 28.6 for 12 ug/seed, decreased slightly to 25.3 for 35 ug/seed, andincreased to 28.9 for 105 ug/seed. These results indicate a potenteffect on water conservation in corn.

Having thus described the basic concept of the invention, it will berather apparent to those skilled in the art that the foregoing detaileddisclosure is intended to be presented by way of example only, and isnot limiting. Various alterations, improvements, and modifications willoccur and are intended to those skilled in the art, though not expresslystated herein. These alterations, improvements, and modifications areintended to be suggested hereby, and are within the spirit and scope ofthe invention. Additionally, the recited order of processing elements orsequences, or the use of numbers, letters, or other designationstherefore, is not intended to limit the claimed processes to any orderexcept as may be specified in the claims. Accordingly, the invention islimited only by the following claims and equivalents thereto.

1. An isolated peptide comprising the amino acid sequence of (i)(SEQ ID NO: 1) (L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X-(L/I/V/F), or (ii) (SEQ ID NO: 2)L-X-X-L-L-L-X-(F/L)-(I/L)-X-X-X-L

wherein X at position 3 is optional and, when present, is any aminoacid; and each X at positions 2, 7, 10, 11, and 12 is any amino acid. 2.The isolated peptide according to claim 1, wherein the peptide is lessthan 100 amino acids in length.
 3. The isolated peptide according toclaim 2, wherein the peptide is between 13 and 50 amino acids in length.4. The isolated peptide according to claim 1, wherein X at position 3 isnot present.
 5. The isolated peptide according to claim 1, wherein X atpositions 3 is present, and is a polar amino acid.
 6. The isolatedpeptide according to claim 1, wherein X at position 2 is a polar aminoacid.
 7. The isolated peptide according to claim 1, wherein X atposition 2 is selected from the group consisting of D, isoD, E, andg-glutamate.
 8. The isolated peptide according to claim 1, wherein thepeptide is free of cysteine and methionine.
 9. The isolated peptideaccording to claim 1, wherein X at position 7 is selected from the groupconsisting of A, M, G, S, or E.
 10. The isolated peptide according toclaim 1, wherein X at position 8 is F or L.
 11. (canceled)
 12. Theisolated peptide according to claim 1, wherein X at position 9 is I orL.
 13. (canceled)
 14. The isolated peptide according to claim 1, whereineach X at positions 10, 11, and 12 is selected independently from thegroup consisting of M, A, G, S, and E.
 15. The isolated peptideaccording to claim 1 further comprising a hydrophilic amino acidsequence at either the N-terminal or C-terminal end of SEQ ID NO:
 1. 16.The isolated peptide according to claim 15, wherein the hydrophilicamino acid sequence comprises from two to ten hydrophilic amino acidresidues.
 17. The isolated peptide according to claim 1, wherein thepeptide comprises the amino acid sequence of: (i) (SEQ ID NO: 3)(Q/E)-(Q/E)-(L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X- (L/I/V/F)-(D/G/Q/E)-(D/G/Q/E),

wherein X at position 5 is optional and, when present, is any aminoacid, and each X at positions 4, 9, 12, 13, and 14 is any amino acid; or(ii) (SEQ ID NO: 4) (L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X-(L/I/V/F)- (D/G/Q/E)-(D/G/Q/E),

wherein X at position 3 is optional and, when present, is any aminoacid, and each X at positions 2, 7, 10, 11, and 12 is any amino acid; or(iii) (SEQ ID NO: 5) (Q/E)-(Q/E)-(L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X- (L/I/V/F), 

wherein X at position 5 is optional and, when present, is any aminoacid, and each X at positions 4, 9, 12, 13, and 14 is any amino acid.18. The isolated peptide according to claim 17, wherein the peptidecomprises the amino acid sequence of: (i) (SEQ ID NO: 3)(Q/E)-(Q/E)-(L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X- (L/I/V/F)-(D/G/Q/E)-(D/G/Q/E),

wherein X at position 5 is optional and, when present, is any aminoacid, and each X at positions 4, 9, 12, 13, and 14 is any amino acid; or(ii) (SEQ ID NO: 5) (Q/E)-(Q/E)-(L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X- (L/I/V/F),

wherein X at position 5 is optional and, when present, is any aminoacid, and each X at positions 4, 9, 12, 13, and 14 is any amino acid.19-28. (canceled)
 29. The isolated peptide according to claim 18,wherein the peptide consists essentially of the amino acid sequence ofSEQ ID NO: 3 or SEQ ID NO:
 5. 30. The isolated peptide according toclaim 17, wherein the peptide comprises the amino acid sequence of:(SEQ ID NO: 4) (L/I/V/F)-X-X-(L/I/V/F)-(L/I/V/F)-(L/I/V/F)-X-(L/I/V/F)-(L/I/V/F)-X-X-X-(L/I/V/F)-(D/G/Q/E)- (D/G/Q/E),

wherein X at position 3 is optional and, when present, is any aminoacid; and each X at positions 2, 7, 10, 11, and 12 is any amino acid 31.The isolated peptide according to claim 1, wherein the peptide comprisesthe amino acid sequence of SEQ ID NOS: 8-26, 28, 29, 31-33, 35-38, 40,41, 46-48, 52, 62, 139, 545, or
 546. 32. The isolated peptide accordingto claim 1, wherein the isolated peptide has an average Kyte-Doolittlehydropathy index of less than 0.7.
 33. The isolated peptide according toclaim 1, wherein the isolated peptide is stable when dissolved in wateror aqueous solution, is resistant to chemical degradation when dissolvedin an aqueous buffer solution containing a biocide, or has a solubilityof greater than about 0.1% in water or aqueous solution. 34-35.(canceled)
 36. The isolated peptide according to claim 1, wherein thepeptide induces an active plant response, but does not induce ahypersensitive response, when applied to plant tissue.
 37. The isolatedpeptide according to claim 36, wherein the peptide comprises the aminoacid sequence of one of P5-6, P5-7, P5-9, P5-10, P5-11, P5-12, P5-13,P5-15, P5-16, P5-17, P5-18, P5-20, P5-24, P5-25, and P5-27 (SEQ ID NOS:140, 13, 15-19, 25-28, 36, and 51-53).
 38. The isolated peptideaccording to claim 1, wherein the peptide induces a hypersensitiveresponse when applied to plant tissue.
 39. The isolated peptideaccording to claim 38, wherein the peptide comprises the amino acidsequence of one of P5, P5a, P5-2, P5-3, P5-4, P5-5, P5-8, P5-14, P5-19,P5-21, P5-22, P5-23, P5-26, P5-28, P5-29, P5-30, P5-31, P5-32, P5-33,and P5-34 (SEQ ID NOS: 8-12, 139, 14, 20, 29, 33, 30, 50, 34, 54, 42,55, 43, 56, 44, and 57).
 40. An isolated peptide comprising the aminoacid sequence of: (SEQ ID NO: 6) J-X-X-J-J-J-X-J-J-X-X-X-J

wherein X at position 3 is optional and, when present, is any aminoacid, and each X at positions 2, 7, 10, 11, and 12 is any amino acid;one to three of the J residues at positions 1, 4, 5, 6, 8, 9, and 13 isa non-hydrophobic amino acid or A, and all other of the J residues areL, I, V, or F; and the peptide induces an active plant response, butdoes not induce a hypersensitive response, when applied to plant tissue.41. The isolated peptide according to claim 40, wherein the peptide isless than 100 amino acids in length.
 42. (canceled)
 43. The isolatedpeptide according to claim 40, wherein not more than one or two of J atpositions 1, 4, 5, 6, 8, 9, and 13 is non-hydrophobic. 44-48. (canceled)49. The isolated peptide according to claim 40, wherein the peptidecomprises the amino acid sequence of P5-35, P5-36, P5-37, P5-38, P5-40,P5-41, P5-45, P5-53, P5-54, P5A single mut 12E, P5A single mut 13E, P5Asingle mut 15E, P5A single mut 16E, and P5A single mut 20E (SEQ ID NOS:71-74, 76, 77, 81, 98-101, 103, 104, 108).
 50. The isolated peptideaccording to claim 1, wherein the peptide is at least 90% pure.
 51. Theisolated peptide according to claim 1 wherein the peptide is a fusionpolypeptide comprising a second amino acid sequence coupled via peptidebond to the amino acid sequence.
 52. The isolated peptide according toclaim 51, wherein the second amino acid sequence includes a purificationtag.
 53. The isolated peptide according to claim 52, wherein the secondamino acid sequence further includes a cleavable linker sequence betweenthe purification tag and the amino acid sequence.
 54. The isolatedpeptide according to claim 51, wherein the peptide is a fusionpolypeptide comprising a first amino acid sequence for said peptidelinked to a second amino acid sequence for said peptide. 55-56.(canceled)
 57. A fusion polypeptide comprising a plurality of amino acidsequences linked together in series, each of the plurality of amino acidsequences comprising the peptide according to claim
 1. 58-59. (canceled)60. A composition comprising one or more peptides according to claim 1,and a carrier.
 61. The composition according to claim 60, wherein thecomposition is a clarified cell extract.
 62. The composition accordingto claim 60 further comprising an additive selected from the groupconsisting of fertilizer, herbicide, insecticide, fungicide, nematicide,a bactericidal agent, a biological inoculant, a plant regulator, andmixtures thereof. 63-67. (canceled)
 68. The composition according toclaim 62, wherein the composition comprises: one or more of peptides P5,P5-8, P5-21, P5-25, P5-27, P5-35, and P5-46 (SEQ ID NOS: 8, 14, 33, 52,53, 71, and 58 respectively); and either (i) clothianidin, a combinationof clothianidin and Bacillus firmus, imidicloprid, or a combination ofimidicloprid and Bacillus firmus; or (ii) thiamethoxam; a combination ofthiamethoxam, mefenoxam, and fludioxynil; a combination of thiamethoxam,mefenoxam, fludioxynil and azoxystrobin; a combination of thiamethoxamand abamectin; a combination of thiamethoxam, abamectin, and a Pasteurianematicide; or a combination of thiamethoxam, mefenoxam, fludioxynil,azoxystrobin, thiabendazole, and abamectin; or (iii) a biologicalinoculant comprising a Bradyrhizobium spp., a Bacillus spp., and acombination thereof. 69-70. (canceled)
 71. The composition according toclaim 60, wherein the carrier is an aqueous carrier optionally furthercomprising one or more of a biocidal agent, a protease inhibitor, anon-ionic surfactant, or a combination thereof.
 72. (canceled)
 73. Thecomposition according to claim 70, wherein the carrier is a solidcarrier in particulate form.
 74. (canceled)
 75. A method of impartingdisease resistance to plants comprising: applying an effective amount ofan isolated peptide according to claim 1 to a plant or plant seed or thelocus where the plant is growing or is expected to grow, wherein saidapplying is effective to impart disease resistance. 76-83. (canceled)84. A method of enhancing plant growth comprising: applying an effectiveamount of an isolated peptide according to claim 1 to a plant or plantseed or the locus where the plant is growing or is expected to grow,wherein said applying is effective to enhance plant growth. 85-92.(canceled)
 93. A method of increasing a plant's tolerance to bioticstress comprising: applying an effective amount of an isolated peptideaccording to claim 1 to a plant or plant seed or the locus where theplant is growing or is expected to grow, wherein said applying iseffective to increase the plant's tolerance to biotic stress factorsselected from the group consisting of insects, arachnids, nematodes,weeds, and combinations thereof. 94-100. (canceled)
 101. A method ofincreasing a plant's tolerance to abiotic stress comprising: applying aneffective amount of an isolated peptide according to claim 1 to a plantor plant seed or the locus where the plant is growing or is expected togrow, wherein said applying is effective to increase the plant'stolerance to abiotic stress factors selected from the group consistingof salt stress, water stress, ozone stress, heavy metal stress, coldstress, heat stress, nutritional stress, and combinations thereof.102-129. (canceled)
 130. A DNA construct comprising a first nucleic acidmolecule encoding a peptide according to claim 1, and apromoter-effective nucleic acid molecule operably coupled to the firstnucleic acid molecule.
 131. (canceled)
 132. A recombinant host cellcomprising the DNA construct according to claim
 130. 133-160. (canceled)